Differentially expressed miR-4310 functions as a tumor suppressor in colorectal cancer

Abstract

BACKGROUND:

Colorectal cancer (CRC) is a digestive tract malignancy microRNAs (miRNAs) have attracted much attention as biomarkers in tumor studies.

OBJECTIVE:

This work focused on the predictive potential and mechanism of miR-4310 in CRC.

METHODS:

The miRNA expression profile sets were obtained from the Gene Expression Omnibus (GEO) database, and the appropriate miRNA was screened by GEO2R. The CRC tissues and control tissues of 88 patients with CRC were collected, and the expression of miR-4310 was detected by quantitative real-time PCR, and the efficacy of miR-4310 in diagnosing CRC was evaluated by the receiver operating characteristic curve (ROC). The effects of miR-4310 on the proliferation, migration, and invasion of CRC cells were explored by Cell Counting Kit-8 (CCK-8) and Transwell experiments. Predicting the potential binding sites of miR-4310 and Runt-related transcription factor 1 (RUNX1) by four predictive websites. The relationship between miR-4310 and RUNX1 was confirmed by a double luciferase reporter gene experiment.

RESULTS:

The bioinformatics analysis found that miR-4310 was differentially expressed in CRC tissues and this finding was certified by the expression of miR-4310 in CRC tissues of collected patients and cultured CRC cell lines. The expression of miR-4310 had a predictive possibility for CRC patients. MiR-4310/RUNX1 pathway had effects on CRC viability, migration, and invasion.

CONCLUSION:

MiR-4310 had the potential to be a biomarker for early screening of CRC. MiR-4310 and RUNX1 participated in the regulation of CRC cells.

Keywords

miR-4310 RUNX1 colorectal cancer biomarker mechanism

1. Introduction

Colorectal cancer (CRC) is located in the colon or rectum, including colon cancer and rectal cancer, which is a common malignant tumor in the digestive system [1]. Patients with CRC have no typical symptoms in the early stage, and the symptoms will become more and more obvious with the gradual development of the tumor, which will spread from local to the whole body [2]. The specific cause of CRC is not clear, which may be caused by the comprehensive effects of personal eating habits, the external environment, and genetic factors [4]. The main treatment methods for CRC are surgical resection, endoscopic submucosal dissection, drug chemotherapy, radiotherapy, and immune-targeted therapy [6]. Despite the continuous development of treatment technology, the incidence and mortality of CRC are still high, and the treatment effect is not satisfactory. Therefore, it is necessary to analyze the potential pathogenesis to help the clinical diagnosis of CRC.

The mechanism of occurrence and development of CRC is complicated. Multiple public papers document that miRNAs not only participate in the proliferation, migration, invasion, and apoptosis of CRC, and also function in tumor angiogenesis, immune system, and tumor-matrix interaction. MiR-21-5p, miR-27b-3p, and miR-15a-3p are involved in the pathogenesis of CRC [7, 8, 9]. MiR-452 is involved in the CRC progression via binding the ERK/MAPK pathway, reflecting its role in the development of CRC [10]. All these publications indicate that several miRNAs were involved in the development of CRC. MiR-4310 is a broadly researched miRNA in tumors, and its effects is revealed in several tumors MiR-4310 is a miRNA that took part in hepatocellular carcinoma, glioma, and lung cancer [11, 12, 13]. MiR-4310 is related to the regulation of lncRNA CASC15 in colon cancer [14]. These previous papers indicated that miR-4310 might be a marker in the development of some malignancies. However, information about miR-4310 on CRC is not elucidated.

In this work, we aimed to study the roles of miR-4310 in CRC. Therefore, the expression and diagnostic role of miR-4310 were evaluated. The impacts of miR-4310 on malignant phenotypes of CRC cells were analyzed.

2. Materials and methods

2.1 Screening of significantly different miRNA

In this study, the miRNA expression profile data sets including the CRC group and control group were screened from Gene Expression Omnibus (GEO) database. Finally, GSE156719 and GSE72281 were selected for the subsequent analysis. After providing and updating the GSE156719 dataset, it contains miRNA sequencing data of 3 tissue samples and 3 paired control tissues from patients. The GSE72281 dataset was submitted and updated, in which we chose miRNA sequencing results from 6 CRC tissues and 6 matched normal mucosa. Differential gene expression analysis was used to identify colorectal tumor samples by using the “GEO2R” interactive tool. The difference of miRNA expression levels between samples was selected by $P<$ 0.05 and $|$ logFC $|$ $>$ 1, then a Venn diagram was drawn to find miRNA with expression difference in tissue for further analysis.

2.2 Participants recruitment

Patients with CRC in People’s Hospital Affiliated to Fujian University of Traditional Chinese Medicine were included. All 88 patients were examined by colonoscopy, and polyethylene glycol electrolyte was used for intestinal cleaning. The inclusion criteria of the patient group were histopathological diagnosis of primary CRC. The exclusion criteria are recurrent CRC, other types of metastatic cancer, pregnant women and lactating women, accompanied by other gastrointestinal diseases, receiving radiotherapy and chemotherapy, and those with cognitive or communication obstacles. CRC tissue and normal tissues were obtained in the surgical resection of tumor tissues. All subjects signed written informed consent and agreed to use their samples. The research plan was reviewed and approved by the Ethics Committee of our hospital.

2.3 Cell foster and transfection

The human CRC cell lines (LOVO, HT29, SW620, Caco-2, and HCT116) and a normal control line (NCM460) are applied in this investigation. All these cell lines were purchased from A.T.C.C (Manassas, America). These cells were cultured in an incubator with 37 C, 95% minimum relative humidity, 5% carbon dioxide content and 95% oxygen content, and grew on the wall. The RPMI 1640 medium was changed every 2 to 3 days on average.

The centrifugal tubes filled with miR-4310-mimic or mimic control (con) dry powder, respectively were obtained from GenePharm (Shanghai, China). The expression vector pcDNA3.1 carrying RUNX1 or empty carriers was purchased from the same company. The Lipofectamine 3000 reagent and sequence to be transfected were mixed, respectively, and then the artificial sequences were transfected into cells with the help of Lipofectamine 3000 for 48 hours.

2.4 Verification of miRNA expression

The total RNA samples were gained from all tissue specimens using Trizol LS reagent (Thermo, San Jose, America). Hifair super mix (YEASEN, Shanghai, China) was prepared for 20 $\mu$ l of reverse transcription reaction system. The reverse transcription PCR tube was put into the PCR instrument and adjusted the parameters to 5 min at 25 ${{}^{\circ}}$ C, 15 min at 55 ${{}^{\circ}}$ C, and 5 min at 85 ${{}^{\circ}}$ C. Under the action of reverse transcriptase, the stem-loop reverse transcription primer finally obtained the reverse transcription product cDNA, which was stored in a refrigerator at $-$ 20 ${{}^{\circ}}$ C. The Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) was used for configuring the PCR reaction system. All specimens need to be tested by PCR three times. The expression level was calculated using the formula, that is, 2^{- ΔΔCT}. The primers of miR-4310 and RUNX1 were designed by Sangon Biotech (Shanghai, China). The mRNA expression of RUNX1 was assessed according to the method of miR-4310 detection.

2.5 Verification of cell normal ability

Firstly, the CCK-8 assay was performed as following steps. A density of 2 $\times$ 10³ cells was seeded to a 96-well plate and the OD data were measured after 0 h, 24 h, 48 h, and 72 h. Before being detected, 10 $\mu$ l CCK-8 (Dojindo, Japan) reagent was added to wells and cultured for 1 h. The absorbance was measured at 450 nm. Secondly, the migrated and invasive abilities were measured using Transwell chambers (Corning, NY, America). The transfected cells with a density of 5 $\times$ 10⁴ and the medium without FBS were added into Transwell upper chambers. The lower chambers were filled with 500 $\mu$ l complete medium. In the invasive assay, the matrigel (BD Biosciences, Franklin Lakes, America) was coated to the upper chambers. The 4% paraformaldehyde was used as a stationary liquid and the 1% crystal violet was used as a dye. Five fields of version were chosen for cell counting.

2.6 Prediction and certification of target gene

The target prediction webs (miRDB, TargetScan, miRTarBase, and DIANA TOOLS) were used to download target genes. The Venn diagram of gene intersection was plotted by VENNY^2.1. The sequence of the target gene was cloned in the pmirGLO carriers as the wide type of RUNX1. The mutant target site was cloned into pmirGLO luciferase reporter vectors. These obtained vectors and the artificial sequences of miR-4310 were transfected into cells. After 48 hours’ post-transfection, the intensity of luciferase in renilla and firefly was detected using the dual luciferase reporter assay system (Promega Corp., Madison, America).

2.7 Statistical analysis

The normal distribution was calculated by Shapiro-Wilk test. Data in accordance with the normal distribution were analyzed by paired Ttest. One-way and two-way ANOVA was used for comparing the discrepancy among three or more groups. The calculations were conducted by IBM SPSS, and the charts are plotted using GraphPad Prism. The clinical predictive possibility of miR-4310 was measured by plotting the receive operator characteristic (ROC) curve. The significant difference was represented by $P<$ 0.05.

3. Results

3.1 Differentially expressed miRNA in CRC

According to the screening criteria of $P<$ 0.05 and $|$ logFC $|$ $>$ 1 in GSE156719, there were 127 differentially expressed miRNAs between CRC and control group (Fig. 1A). In GSE72281, there were 177 differentially expressed miRNAs between CRC and control group (Fig. 1B). After plotting Venn, seventeen kinds of miRNAs (miR-20a-3p, miR-139-5p, miR-1909-3p, miR-4310, miR-196b-5p, miR-135b-5p, miR-1244, miR-92a-3p, let-7d-3p, miR-4687-3p, miR-17-5p, miR-483-5p, miR-93-5p, miR-3162-5p, miR-21-3p) were screened (Fig. 1C). Among all these miRNAs, the intersection gene miR-4310 was obtained for subsequent research.

Figure 1.

The differentially expressed miRNAs in CRC. (A) Differentially expressed miRNAs of GSE156719. (B) Differentially expressed miRNAs in GSE72281. (C) The Venn diagram was plotted using the data from GSE156719 and GSE72281.

Figure 2.

The relative expression of miR-4310 and its potential to be a diagnostic biomarker. (A) The expression of miR-4310 was decreased in CRC tissues. (B) The high AUC value of the ROC curve showed that miR-4310 might act as a diagnostic indicator. ^***P < 0.001, compared to control tissue.

Figure 3.

The effect of miR-4310 on CRC cells. (A) The decreased miR-4310 was observed in CRC cell lines. (B) Transfection of mimics into LOVO and HT29 cells increased the expression of miR-4310. (C–D) Increased miR-4310 expression inhibited cell viability. (E) The migratory cells were suppressed by the overexpression of miR-4310. (F) Upregulation of miR-4310 inhibited the invasion of CRC cells. ^**P < 0.01, ^***P < 0.001, compared to the mimic-con group.

Figure 4.

Target gene of miR-4310. (A) The Venn diagram of predicted targets. (B) Bioinformatics analysis predicted the targeted sequence. (C) The reduction of luciferase activity proved the interaction of RUNX1 with miR-4310. (D) Increased miR-4310 restricted the expression of RUNX1. ^***P < 0.001, compared to the mimic-con group.

Figure 5.

RUNX1/miR-4310 participated in the bioactivity of CRC cells. (A) The expression of RUNX1 was reduced in miR-4310 mimic group and its expression was reversed by the transfection of p-RUNX1. (B–C) The inhibited cell viability was reversed by the upregulation of RUNX1. (D–E) RUNX1 affected the suppressed function of miR-4310 on cell migration and invasion. ^**P < 0.01, ^***P < 0.001, compared to the mimic-con group; ^#P < 0.05, ^###P < 0.001, compared to the miR-4310 mimic $+$ p-RUNX1 group.

3.2 Clinical predictive possibility in CRC

As expressed in Fig. 2A, we observed that circulating miR-4310 was remarkably lower in CRC tissues compared with control tissues.

Then, the value of miR-4310 in diagnosing CRC was evaluated by ROC analysis (Fig. 2B). MiR4310 had a high efficiency in screening CRC, with an AUC value of 0.908, a specificity of 0.830 and a sensitivity of 0.841. It can be seen from the above results that miR-4310 could effectively predict CRC, and could be used as new markers for CRC screening.

3.3 The impacts of miR-4310 on CRC cell lines

The decreased miR-4310 expression was observed in all CRC cells compared with NCM460 cells (Fig. 3A, $P<$ 0.001). Comparing the discrepancy of miR-4310 in all CRC cells, the LOVO and HT29 were chosen for the following experiments. The concentration of miR-4310 in the transfection of the miR-4310 mimic group was elevated in both LOVO and HT29 cells (Fig. 3B, $P<$ 0.001). The CCK-8 assay documented that the viability of CRC cell lines was ameliorated by the overexpression of miR-4310, indicating that miR-4310 might associate with the viability of CRC cells (Fig. 3C and D, $P<$ 0.01). The number of migratory cells in the miR-4310 mimic group was decreased in two types of CRC cells, suggesting that miR-4310 took part in the migration of CRC cells (Fig. 3E, $P<$ 0.001). The upregulation of miR-4310 inhibited the number of invaded cells in LOVO and HT29 cells, which was similar to the results of migration experiments (Fig. 3F, $P<$ 0.001).

3.4 Target gene of miR-4310 in CRC

The intersection of target genes of miR-4310 in four websites was shown in Fig. 4A. The binding site between miR-4310 and RUNX1 was shown in Fig. 4B. The relative luciferase activity was suppressed in the group with miR-4310 mimic and wide-type RUNX1 in HT29 cells and LOVO cells (Fig. 4C, $P<$ 0.001), while there was no prominent difference in the relative content of luciferase between mutant RUNX1 plasmid and miR-4310 mimics co-transfection group and mutant RUNX1 plasmid and control mimics co-transfection group (Fig. 4C, $P>$ 0.05), reflecting that RUNX1 might target miR-4310. The increased miR-4310 expression in the LOVO and HT29 cells decreased the mRNA expression of RUNX1 (Fig. 4D, $P<$ 0.001).

3.5 RUNX1 participated in the antitumor effects of miR-4310

The transfection effect of this overexpression plasmid was shown in Fig. 5A, and in this picture, the mRNA expression of RUNX1 was inhibited by the increased expression of miR-4310 and improved by the cotransfection of miR-4310 mimics and p-RUNX1 ( $P<$ 0.001). The suppressed cell viability in the miR-4310 mimic group was reversed by the overexpressed RUNX1 (Fig. 5B and C, $P<$ 0.05). The migratory and invaded cells were repressed by increased miR-4310 expression, which was reversed by the enhanced levels of RUNX1 (Fig. 5D and E, $P<$ 0.001).

4. Discussion

In the current study, we screened differently expressed miRNAs in patients with CRC. Then, the expression and diagnostic possibility of miR-4310 in patients with CRC were estimated. Finally, the potential effects of miR-4310 on CRC cells were researched. The expression of miR-4310 showed a reduction in CRC tissues and diagnostic value for patients with CRC. Overexpression of miR-4310 inhibited the viability, migration, and invasion of CRC cells by targeting RUNX1.

With the continuous innovation of clinical diagnosis and treatment technology, the mortality rate of CRC patients has effectively reduced, but there are still some defects in the existing CRC screening measures [15]. Scholars provide new insights for the screening of patients with CRC and the pathogenesis of CRC with the in-depth study of miRNA [16, 17]. In a clinical study of CRC, miR-99a, miR-1246, miR-1299, and miR-1539 are abnormally expressed in patients with CRC, and their possible predictive roles for distinguishing CRC patients are researched [18, 19, 20]. With the improvement of miRNA sequencing technology, scientists’ knowledge of malignant has been greatly promoted [21]. More and more public genome databases, such as the GEO, are commonly used to research potential tumor diagnostic markers [22]. In this study, firstly, we screened the abnormally expressed miRNAs in the tissues of CRC from the profiles GSE156719 and GSE72281. Then, we assessed the expression levels of circulating miR-4310 in tumor and control tissues from patients with CRC by qRT-PCR and analyzed their values as screening markers of CRC by ROC. Finally, the function and potential mechanism of miR-4310 in CRC cell lines were explored.

In this work, our team analyzed the data of GSE156719 and GSE72281 according to the data of CRC tissues and control tissues through NCBI online analysis software GEO2R and Venn. Finally, it was concluded that the expression of miR-4310, an obviously different miRNA, decreased in CRC tissues from patients. In different tumors, miR-4310 may play a role as a tumor suppressor or carcinogen. Previous studies showed that the levels of miR-4310 were declined in patients with hepatocellular carcinoma [12]. But in the tissues from glioma, Wu et al. report that the content of miR-4310 is overexpressed compared to the non-tumor brain tissues, underlining that miR-4310 might function as an activator in the carcinogenesis of glioma [13]. The main reasons causing this may be contributed to the different mechanisms of malignant progression in tissues. In this work, we observed that the expression level of miR-4310 in CRC tissue was obviously decreased, indicating miR-4310 acted as a tumor inhibitory role in CRC. Furthermore, miR-4310 could screen CRC patients with high sensitivity and specificity through the analysis of ROC.

Recent papers find that miRNAs are not only involved in tumorigenesis and development but also regulate the expression of oncogenes or suppressor indicators MiR-139-5p, miR-135b, and miR-93 are involved in the development and mechanism of CRC [23, 24, 25]. MiR-503-5p influences the phenotype of CRC cells by targeting SALL1, thus participating in the progression of CRC [26]. Yu et al. report that miR-17-5p has a suppressive effect on CRC cells’ normal abilities, and the rescue assays indicate that HAPB2 reversed the impacts of MiR-17-5p on CRC [27]. In this work, we verified that the expression of miR-4310 in CRC cell lines obviously declined, which was similar to the concentration of CRC tissues. To clarify the biological influence of miR-4310, we transfected miR-4310 mimics into LOVO cells and HT29 cells respectively and verified that the mimics could effectively increase the content of miR-4310 in the two cell lines, which indicated the feasibility of transfection experiment. CCK-8 experiment was used to explore the effect of miR-4310 on the activity of tumor cells. The results showed that miR-4310 mimics can effectively reduce the activity of tumor cells, reflecting that miR-4310 may influence the growth of CRC. Migration and invasion ability is an important biological phenotype of malignant tumors, so we used the Transwell experiment to explore the influence of miR-4310 on the migration and invasion ability of CRC cells. Overexpression of miR-4310 inhibited the migration and invasion ability of CRC cells. Hence, miR-4310 is a tumor suppressor of CRC, which can effectively inhibit the activity, migration and invasion of tumor cells, and may become a potential target of CRC drug therapy in the future.

Four predictive websites were performed to forecast the downstream targets of miR-4310, and through the intersection of all findings, we choose RUNX1 for the following study. According to the statement of an article of 2019, the expression of RUNX1 was elevated and it played roles in the exacerbation and metastasis process [28]. Lu et al. also underline that RUNX1 is upregulated in CRC tumor tissues compared to normal epithelial tissues and affects the migration of CRC cells, indicating that RUNX1 is associated with the mechanism of CRC [29]. The results of the current work suggested that overexpression of miR-4310 can significantly reduce the luciferase activity of wild-type RUNX1, but has no obvious effect on the luciferase activity of mutant RUNX1. In addition, we up-regulated the contents of miR-4310 and then detected the expression of RUNX1 gene. The results suggest that the overexpression of miR-4310 reduced the expression of RUNX1 in CRC cells. In in vitro function acquisition experiments, the miR-4310 could inhibit cell viability, migration, and invasion, while the increased RUNX1 expression reversed the function of miR-4310 on CRC cells, indicating that miR-4310/RUNX1 was correlated to the mechanism of CRC development. Limitations exist in this current study, Firstly, this work did not include patients with other intestines problems, for instance inflammatory bowel diseases or colorectal adenomas, and analyze the prognostic possibility of miR-4310. Secondly, the small sample size of included CRC patients might decrease the reliability of diagnostic analysis of miR-4310. Further study with a larger sample size needs perform to certified that whether miR-4310 expression can be used in normal clinical diagnosis.

5. Conclusion

The miR-4310 was screened through GEO database and we found that it was downexpressed in CRC tissues according to the GEO2R analysis. Based on our certification in patients with CRC, the expression of miR-4310 was decreased in the CRC tissues and it had the potential to be a biomarker for diagnosing CRC. MiR-4310 might function as a tumor suppressor in CRC by ameliorating the viability, migration, and invasion of CRC cells. RUNX1 is a downstream target participating in the regulation of miR-4310 in CRC.

Footnotes

Acknowledgments

The authors have no acknowledgments.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

The authors report no funding.

References

Wang

Zhou

Hua

Zhu

Liu

. CDK3, CDK5 and CDK8 proteins as prognostic and potential biomarkers in colorectal cancer patients. Int J Gen Med. 2022; 15: 2233-45. doi: 10.2147/ijgm.s349576.

Chen

. DUSP4 directly deubiquitinates and stabilizes Smad4 protein, promoting proliferation and metastasis of colorectal cancer cells. Aging (Albany NY). 2020; 12(17): 17634-46. doi: 10.18632/aging.103823.

Dekker

Tanis

Vleugels

JLA

Kasi

Wallace

. Colorectal cancer. Lancet. 2019; 394(10207): 1467-80. doi: 10.1016/s0140-6736(19)32319-0.

Yeom

Lim

. Effects of the enhanced recovery after surgery (ERAS) program for colorectal cancer patients undergoing laparoscopic surgery. Clin Nutr Res. 2022; 11(2): 75-83. doi: 10.7762/cnr.2022.11.2.75.

Jiang

Fan

Tao

, et al. Association between physical activity and cancer risk among Chinese adults: A 10-year prospective study. Int J Behav Nutr Phys Act. 2022; 19(1): 150. doi: 10.1186/s12966-022-01390-1.

Yin

Guo

. HOXD13 promotes the malignant progression of colon cancer by upregulating PTPRN2. Cancer Med. 2021; 10(16): 5524-33. doi: 10.1002/cam4.4078.

Sun

Zhou

Han

Liu

Zhang

, et al. The c-Myc/miR-27b-3p/ATG10 regulatory axis regulates chemoresistance in colorectal cancer. Theranostics. 2020; 10(5): 1981-96. doi: 10.7150/thno.37621.

Jiang

Chen

Wang

Liu

Chen

, et al. MiR-21-5p induces pyroptosis in colorectal cancer via TGFBI. Front Oncol. 2020; 10: 610545. doi: 10.3389/fonc.2020.610545.

Liu

Yao

Zhou

Chen

Shi

, et al. MiR-15a-3p regulates ferroptosis via targeting glutathione peroxidase GPX4 in colorectal cancer. Mol Carcinog. 2022; 61(3): 301-10. doi: 10.1002/mc.23367.

10.

Lin

Han

Lai

, et al. MiR-452-5p promotes colorectal cancer progression by regulating an ERK/MAPK positive feedback loop. Aging (Albany NY). 2021; 13(5): 7608-26. doi: 10.18632/aging.202657.

11.

Chakraborty

Nath

. A study on microRNAs targeting the genes overexpressed in lung cancer and their codon usage patterns. Mol Biotechnol. 2022; 64(10): 1095-119. doi: 10.1007/s12033-022-00491-3.

12.

Chen

Zhang

Yuan

Liu

Ding

, et al. MiR-4310 regulates hepatocellular carcinoma growth and metastasis through lipid synthesis. Cancer Lett. 2021; 519: 161-71. doi: 10.1016/j.canlet.2021.07.029.

13.

Luo

Huang

Ding

Xie

, et al. MiR-4310 induced by SP1 targets PTEN to promote glioma progression. Cancer Cell Int. 2020; 20(1): 567. doi: 10.1186/s12935-020-01650-9.

14.

Jing

Huang

Guo

Yang

Chen

, et al. LncRNA CASC15 promotes colon cancer cell proliferation and metastasis by regulating the miR-4310/LGR5/Wnt/β-catenin signaling pathway. Mol Med Rep. 2018; 18(2): 2269-76. doi: 10.3892/mmr.2018.9191.

15.

Zhao

Zhou

Yin

Zhou

Wang

, et al. Circulating miR-627-5p and miR-199a-5p are promising diagnostic biomarkers of colorectal neoplasia. World J Clin Cases. 2022; 10(16): 5165-84. doi: 10.12998/wjcc.v10.i16.5165.

16.

Huang

Zhu

Jin

Zhang

, et al. Dissecting miRNA signature in colorectal cancer progression and metastasis. Cancer Lett. 2021; 501: 66-82. doi: 10.1016/j.canlet.2020.12.025.

17.

Zanutto

Ciniselli

Belfiore

Lecchi

Masci

Delconte

, et al. Plasma miRNA-based signatures in CRC screening programs. Int J Cancer. 2020; 146(4): 1164-73. doi: 10.1002/ijc.32573.

18.

Zhu

Liu

Lin

Cai

, et al. Influence and mechanism of miR-99a suppressing development of colorectal cancer (CRC) with diabetes mellitus (DM). Onco Targets Ther. 2019; 12: 10311-21. doi: 10.2147/ott.s190998.

19.

Rafiee

Razmara

Motavaf

Mossahebi-Mohammadi

Khajehsharifi

Rouhollah

, et al. Circulating serum miR-1246 and miR-1229 as diagnostic biomarkers in colorectal carcinoma. J Cancer Res Ther. 2022; 18(Supplement): S383-s90. doi: 10.4103/jcrt.JCRT_752_20.

20.

Cui

Ding

Xing

Yuan

. MiR-1539 and its potential role as a novel biomarker for colorectal cancer. Front Oncol. 2020; 10: 531244. doi: 10.3389/fonc.2020.531244.

21.

Wen

Britton

Garje

Darbro

Packiam

. The emerging role of somatic tumor sequencing in the treatment of urothelial cancer. Asian J Urol. 2021; 8(4): 391-9. doi: 10.1016/j.ajur.2021.06.005.

22.

Wang

Shi

Zhang

Zhou

Huang

Zhou

, et al. PRKCB is relevant to prognosis of lung adenocarcinoma through methylation and immune infiltration. Thorac Cancer. 2022; 13(12): 1837-49. doi: 10.1111/1759-7714.14466.

23.

Zhang

Dong

Zhu

Tsoi

Zhao

, et al. microRNA-139-5p exerts tumor suppressor function by targeting NOTCH1 in colorectal cancer. Mol Cancer. 2014; 13: 124. doi: 10.1186/1476-4598-13-124.

24.

Jia

Luo

Ren

Liu

, et al. miR-182 and miR-135b mediate the tumorigenesis and invasiveness of colorectal cancer cells via targeting ST6GALNAC2 and PI3K/AKT pathway. Dig Dis Sci. 2017; 62(12): 3447-59. doi: 10.1007/s10620-017-4755-z.

25.

Chen

Liu

Zhang

Liu

Cheng

Zhang

, et al. Exosome-mediated transfer of miR-93-5p from cancer-associated fibroblasts confer radioresistance in colorectal cancer cells by downregulating FOXA1 and upregulating TGFB3. J Exp Clin Cancer Res. 2020; 39(1): 65. doi: 10.1186/s13046-019-1507-2.

26.

Wang

. Ultrasound microbubble-mediated miR-503-5p downregulation suppressed in vitro CRC progression via promoting SALL1 expression. Tissue Cell. 2022; 76: 101811. doi: 10.1016/j.tice.2022.101811.

27.

Wang

Xuan

Han

Zhang

, et al. miR-17-5p promotes the invasion and migration of colorectal cancer by regulating HSPB2. J Cancer. 2022; 13(3): 918-31. doi: 10.7150/jca.65614.

28.

Lai

Fang

Yan

Zhang

, et al. RUNX1 promotes tumour metastasis by activating the Wnt/β-catenin signalling pathway and EMT in colorectal cancer. J Exp Clin Cancer Res. 2019; 38(1): 334. doi: 10.1186/s13046-019-1330-9.

29.

Yang

Lin

Cai

. RUNX1 regulates TGF-β induced migration and EMT in colorectal cancer. Pathol Res Pract. 2020; 216(11): 153142. doi: 10.1016/j.prp.2020.153142.