The new cases of GC in China account for about 50% of the total globally 14, most patients have already been in the locally advanced or advanced stage at the time of first diagnosis, and the median survival time of patients with metastatic GC is only 8–10 months 15. Molecular targeted therapy has attracted increasingly more attention due to its specific anti-cancer effect, but it relies on more useful molecular targets. Only 12–15% of patients with metastatic GC are positive for HER-2. The anti-HER-2 monoclonal antibody trastuzumab combined with chemotherapy can be used as the first-line treatment 16. However, there has been no targeted therapy for most metastatic GC. GC patients in China are greatly different from those in European and American countries in the clinical stage, site of primary foci, and recurrence and metastasis patterns. Therefore, it is of important clinical significance to explore molecules related to GC metastasis in Chinese patients at the molecular biological level. Besides, tumor diagnosis is an important process that may affect the tumor progression, and the risk of recurrence and death, so it is necessary to explore early detection or screening methods for a variety of cancers. Therefore, biomarkers for cancer detection and progression have been explored in multiple studies.
More than 2500 miRNAs (miRBase database) have been identified so far, and their ectopic expressions are associated with tumor proliferation, invasion, metastasis, growth and apoptosis. There are abnormal expressions of many miRNAs in the plasma and serum of GC patients. For instance, miR-212, miR-423, miR-215, miR-131, miR-988-5p and miR-301-3p have overexpression in the serum of GC patients 17. Carcinogenic miRNAs and tumor suppressor miRNAs are important players in the progression of GC. For example, restoring miR-34 obviously inhibits GC growth and metastasis through promoting the expression of tumor suppressor mutant p53 18. In this study, it was found by bioinformatics analysis that miR-185-5p was lowly expressed in GC, consistent with previous reports 19. Besides, SMAD3 was highly expressed in GC, leading to a low survival rate. The transcription factor SMAD3 is a central regulator in the downstream of the TGF-β1/Smad signaling pathway, and it is involved in regulating various physiological and pathological processes, one of which is carcinogenesis. At the same time, the regulatory effects of miR-185-5p on the proliferation, migration and invasion of GC cells were detected by in vitro experiments.
There is a study showing that down-regulated miR-185-5p in non-small cell lung cancer is correlated with poorer cell differentiation and later lymph node stage 13, indicating that miR-185-5p may be implicated in the growth and metastasis of primary tumors. The specific regulatory mechanism, however, may vary among tumor tissues. In this study, the opposite expression levels of miR-185-5p and SMAD3 protein were found in GC, and the SMAD3 protein expression significantly declined after HGC-27 cells were transfected with miR-185-5p mimic. The above findings demonstrate that miR-185-5p may inhibit migration and invasion of GC cells by targeting SMAD.
Therefore, the indexes related to proliferation, migration and invasion of GC cells were further detected in this study. The results showed that miR-185-5p could significantly lower the expression levels of migration- and invasion-related proteins MMP2, MMP3 and MMP9, and proliferation-related proteins Cyclin D1, PCNA and P21. MMPs are zinc-containing enzymes capable of degrading all extracellular matrix components, which is an important event during invasion and metastasis of most tumor cells 20, and they may be implicated in the growth, differentiation, migration, apoptosis, invasion and metastasis of cancer cells 21. Therefore, the down-regulation of MMP2, MMP3 and MMP9 protein expressions in HGC-27 cells in miR-mimic group indicates that miR-185-5p can inhibit the migration and invasion of GC cells, which was also confirmed by wound healing assay and Transwell assays. Cyclin D1 is an important gene of the cell cycle, which mainly acts in the G1 phase and is an important player in cell cycle regulation. The high expression of Cyclin D1 can facilitate cell proliferation and differentiation, which is closely related to tumorigenesis and development 22. It has been found that the level of Cyclin D1 has close correlations with tumor stage, degree of differentiation and lymphatic metastasis, and it serves as an important index for the grade of malignancy 23. Meanwhile, cell proliferation and differentiation are regulated by related proteins. Currently, PCNA 24 and cyclin-dependent kinase inhibitor family member P21 25 are widely-studied proteins that regulate cell proliferation. The higher the PCNA index, the quicker the cell division and proliferation. Ultimately, the cells will gain the ability of infinite proliferation, and their morphology and structural function will be altered, thus promoting cell proliferation 24. P21 is a cell cycle regulatory protein able to arrest cells in the G1 phase, promote self-repair of mildly damaged DNA, or make heavily damaged DNA enter the apoptotic process to prevent abnormal mutations 25. In this study, the protein levels of Cyclin D1, PCNA and P21 in GC cells significantly rose after low expression of miR-185-5p in vitro, which, however, was reversed after overexpression of miR-185-5p. Moreover, after HGC-27 cells were transfected with miR-185-5p mimic, their viability and the number of colonies formed significantly declined. It can be inferred that overexpression of miR-185-5p can suppress the proliferation of GC cells(Fig. 6).
In conclusion, miR-185-5p can suppress the growth, invasion and migration of HGC-27 cells via targeting SMAD3, whose mechanism is related to the proteins that regulate cell growth, invasion and migration. However, there are many signaling pathways regulating the growth, invasion and migration of HGC-27 cells. In subsequent experiments, the effects of miR-185-5p targeting SMAD3 on the growth, invasion and migration signaling pathways of HGC-27 cells can be further investigated.