TRPM7 deficiency suppresses cell proliferation,migration,and invasion in human colorectal cancer via regulation of epithelial-mesenchymal transition

Abstract

BACKGROUND:

It has been documented that transient receptor potential melastatin 7 (TRPM7) plays a pivotal role in the development of multiple cancers. However, the role of TRPM7 in human colorectal cancer (CRC) is poorly understood. Therefore, the aim of this study was to investigate the expression and significance of TRPM7 in CRC.

METHODS:

In this study, TRPM7 expression was first investigated in Gene Expression Omnibus (GEO), and then validated it with the data from our medical center. CCK-8, colony survival, transwell, and flow cytometry assays were employed to evaluate the effects of TRPM7 knockdown on the CRC cell proliferation, migration, and invasion, as well as cell cycle and apoptosis.

RESULTS:

We observed markedly increased TRPM7 expression in CRC tissues. CRC patients with high expression of TRPM7 suggested deeper tumor infiltration, positive lymph node metastasis, distant metastasis, and advanced clinical stage. In addition, TRPM7 was also overexpressed in CRC cell lines. Downregulated TRPM7 in vitro suppressed CRC cell proliferation, migration, and invasion, as well as triggered cell cycle arrest at the G0/G1 phase, reduced the S phase, and promoted apoptosis. Importantly, decreased TRPM7 in CRC cells reversed the epithelial-mesenchymal transition (EMT) status, accompanied by downregulation of N-cadherin and upregulation of E-cadherin.

CONCLUSION:

Our study indicated that the expression of TRPM7 was positively correlated with tumor infiltration, lymph node metastasis, distant metastasis and clinical stage of CRC. Besides, decreased TRPM7 in vitro inhibited CRC cell proliferation, migration and invasion by modulating EMT.

Keywords

TRPM7 colorectal cancer proliferation migration invasion epithelial-mesenchymal transition

1. Introduction

Colorectal cancer (CRC) is one of the leading causes of cancer-associated mortality, resulting in a heavy healthcare burden in China [1]. Although the survival of patients with clinically localized CRC has been steadily enhanced by effective surgical and chemotherapeutic treatments, the prognoses of those with metastatic CRC remain unsatisfactory [2]. The alteration of oncogenes and tumor suppressor genes mainly initiates multiple biological events involved in CRC progression [3]. Thus, it is of great significance to comprehensively understand the molecular mechanisms underlying the pathogenesis of CRC.

Transient receptor potential melastatin (TRPM) is a superfamily of cation-selective channels located on the cell membrane, consisting of eight members, namely TRPM1 to TRPM8 [4]. In our previous study, gene expression microarray analysis was used to analyze differentially expressed genes (DEGs) between CRC and paired adjacent normal tissues (PANTs), which revealed high expression levels of the TRPM7 gene in colorectal cancer (CRC) [5]. TRPM7 is a bifunctional ion channel with a functional serine/threonine-specific protein kinase domain, exhibiting abundant expression in various human cells [6]. As a protein kinase enzyme, TRPM7 phosphorylates itself and other downstream substrates. The activity of TRPM7 is important for the function of ion channels and various cellular processes, including growth, migration, and survival. As an ion channel, it is highly permeable to both divalent (Ca ${}^{2+}$ /Mg ${}^{2+}$ ) and monovalent cations, exerting a great impact on human Ca ${}^{2+}$ /Mg ${}^{2+}$ metabolism [7]. Recently, accumulating evidence has shown that TRPM7 is related to the proliferation, migration, and invasion of tumor cells [8, 9, 10, 11, 12].

Epithelial-mesenchymal transition (EMT) is a biological process comprising the trans-differentiation of epithelial cells to mesenchymal cells. Notably, EMT is pathologically involved in fibrosis and carcinogenesis. During EMT, cells gradually relinquish their epithelial characteristics and, simultaneously, acquire mesenchymal properties; this process is characterized by decreased expression of E-cadherin and increased expression of N-cadherin [13, 14, 15]. Additionally, EMT enables cell motility and invasiveness. Considerable research has demonstrated EMT as a critical step for invasion and metastasis in various cancers, including CRC [13, 16, 17].

To this end, the expression of TRPM7 was first examined in the Gene Expression Omnibus (GEO) database, followed by further confirmation at our institute. Subsequently, TRPM7 expression was assessed in clinical samples, followed by an investigation of the relationship between TRPM7 expression and clinicopathological factors. Moreover, we evaluated the effects of TRPM7 on proliferation, migration, and invasion in CRC cells, to further assess the involvement of EMT in these processes.

2. Materials and methods

2.1 Microarray data processing

Microarray datasets of CRC, including GSE41328, GSE39582, and GSE32323, were downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). Robust Multichip Average (RMA), used for the adjustment of the background in raw files, is an algorithm for the creation of expression matrixes from Affymetrix data. The limma package (http://bioconductor.org/ packages/release/bioc/html/limma.html) was used to select DEGs [18]. Adjusted $P<$ 0.01 and $|$ log2 fold change (FC) $|\geqslant$ 2.0 of genes were used as the cut-off criteria for screening DEGs. Consistent with the outcomes of our microarray analysis [5], among all DEGs, the expression of the TRPM7 gene was enhanced in CRC tissues. Therefore, we further investigated the functional roles of TRPM7.

Table 1
List of primary antibodies

Antibodies	Dilution (WB)	Dilution (IF)	Supplier
Anti-TRPM7	1:500	1:100	Abcam, UK, Cat. #ab109438
Anti-E-cadherin	1:1000	1:100	Cell signaling technology, USA, Cat. #3195
Anti-N-cadherin	1:1000	1:100	Cell signaling technology, USA, Cat. #13116
Anti-GAPDH	1:10000	–	Sungene biotech, china, KM9002T

2.2 Human CRC tissues

This study was strictly performed according to the Declaration of Helsinki; it was approved by the Research Ethics Committee of Zhongnan Hospital of Wuhan University (approval number: 201511). All CRC patients provided written informed consent after sufficient communication. Clinical samples were obtained from patients who had been pathologically diagnosed with CRC and underwent surgery at Zhongnan Hospital of Wuhan University.

The tissue microarray was prepared with six pairs of CRC tissue and PANT (more than 5 cm away from the cancer tissue) collected after surgery that took place between June 2012 and November 2012 [5]. A total of 103 CRC patients underwent surgical resection between January 2015 and June 2016; subsequently, their clinicopathological data were collected.

2.3 Cell culture

Human CRC cell lines HT29, DLD1, SW480, LOVO, and HCT116 and the human normal intestinal mucosa cell line NCM460 were commercially obtained from the Cell Center of the Chinese Academy of Sciences (Shanghai, China). The NCM460, SW480, HT29, DLD1, and HCT116 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM); the LOVO cells were cultured in Ham’s F-12K (Kaighn’s) medium at 37 ${}^{\circ}$ C in a humidified incubator containing 5% CO ${}_{2}$ . Both types of media were supplemented with 10% fetal bovine serum (FBS) (Gibco, Australia).

2.4 RNA isolation and RT-qPCR

RNA isolation and subsequent RT-qPCR were conducted in line with previously described methods [19]. The primer sequences for TRPM7 were: 5 ${}^{\prime}$ -TGGATG ATGGCACTGTTGGAA-3 ${}^{\prime}$ (sense) and 5 ${}^{\prime}$ -CATTTGG CCCACCCTCAAATATAA-3 ${}^{\prime}$ (antisense). The primer sequences for the $\beta$ -actin housekeeping gene (our internal control) were: 5 ${}^{\prime}$ -AGAGCTACGAGCTGCCTG AC-3 ${}^{\prime}$ (sense) and 5 ${}^{\prime}$ -AGCACTGTGTTGGCGTACA G-3 ${}^{\prime}$ (antisense). The RT-qPCR reaction was performed as follows; pre-denaturation: 95 ${}^{\circ}$ C for 5 min; amplification (40 cycles): 95 ${}^{\circ}$ C for 30 s, 60 ${}^{\circ}$ C for 30 s, and 72 ${}^{\circ}$ C for 30 s. The 2 ${}^{\rm-\Delta\Delta CT}$ method was used to calculate the mRNA expression of TRPM7 after normalization to $\beta$ -actin [20].

2.5 siRNA transfection

siRNA was transfected into the TRPM7-expressing cell lines HT29 and SW480 using lipoJetTM (SignaGen, China) in line with standard protocols. TRPM7-specific siRNA (siTRPM7) and control siRNA (siCON) were synthesized by GenePharma (Shanghai, China). The siTRPM7 sequence was 5 ${}^{\prime}$ -GCGCUUUC CUUAUCCACUUTT-3 ${}^{\prime}$ , whereas the siCON sequence was 5 ${}^{\prime}$ -UUCUCCGAACGUGUCACGUTT-3 ${}^{\prime}$ . After siRNA transfection for 48 h, RT-qPCR and western blot analyses were performed to determine the transfection efficiency.

2.6 Cell viability

Cell viability was analyzed using the Cell Counting Kit-8 (CCK-8 kit, Dojindo, Japan) in line with previously described methods [17].

2.7 Clonogenic survival

A clonogenic survival assay was conducted as previously described [19].

2.8 Flow cytometry

Cell cycle and apoptosis assays were conducted via flow cytometry in accordance with previous protocols [19].

2.9 Transwell migration and invasion assays

Transwell chambers (Corning, USA), with an 8.0- $\mu$ m pore size, were used to perform migration and invasion assays. After transfection for 48 h, CRC cells were adjusted at a density of 1 $\times$ 10 ${}^{6}$ cells/mL and suspended in serum-free medium. For the Transwell migration assay, 200 $\mu$ L of the CRC cell suspension was added to the top chamber. DMEM containing 10% FBS was added to the lower chamber, which was placed in a 24-well plate. After incubation at 37 ${}^{\circ}$ C for 24 h, a cotton swab was used to wipe cells from the upper chamber; the migrated cells on the lower surface of the membrane were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. Subsequently, the number of migrated cells was counted and analyzed from eight random views using a microscope. For the Transwell invasion assay, the upper surface of the Transwell chamber was pre-coated with 40 $\mu$ L Matrigel (1 mg/mL; Sigma-Aldrich, USA) and placed in a 37 ${}^{\circ}$ C incubator overnight. Subsequently, 100 $\mu$ L of the CRC cell suspension was inoculated into the upper chamber of each well and placed in a 24-well plate, after which DMEM containing 10% FBS was added to the lower chamber of each well. For both assays, the cells were fixed, stained, and counted after incubation for 48 h at 37 ${}^{\circ}$ C.

Figure 1.

Increase of TRPM7 expression in GEO and TCGA databases in CRC. (A) DEGs in CRC tissues vs PANTs across each independent dataset. Each column represents a sample and each row represents the expression level of a given mRNA. The color scale represents the raw Z score ranging from blue (low expression) to red (high expression). (B) Venn diagrams of overlapping DEGs between GSE32323, GSE39582 and GSE41328.

2.10 Western blot analysis

Total proteins were extracted from CRC tissues and cells using a RIPA lysis buffer containing a protease inhibitor (Sigma-Aldrich, USA). The specific procedure followed for the western blot analysis was the same as that used in our previous study [19]. The primary antibodies are listed in Table 1, and the appropriate horseradish peroxidase-labeled IgG secondary antibodies (1:10000, LK2001 or LK2003, Sungene Biotech, China) were used. The Enhanced Chemiluminescence kit (Bio-Rad, USA) was used to visualize the protein bands, followed by quantification with ImageJ software (version 1.46; NIH, Bethesda, MD, USA).

2.11 Immunofluorescence staining

Immunofluorescence (IF) staining was performed in line with standard protocols. CRC tissue specimens were fixed with 4% PFA at 4 ${}^{\circ}$ C overnight and subsequently embedded in paraffin, using a tissue processor (Thermo Fisher Scientific, USA); 4 $\mu$ m-thick sections were then obtained using a rotation microtome (Thermo Fisher Scientific). Thereafter, the sliced sections were labeled with primary antibodies (shown in Table 1) and, subsequently, with a Cy3-labeled IgG secondary antibody (1:100, BA1032, Boster Biological Technology, China) at room temperature. Finally, 4’,6-diamidino-2-phenylindole (DAPI) was used for staining the nuclei, and images were obtained with a fluorescence microscope (Olympus, Japan).

Figure 2.

High expression of TRPM7 validated by our own database in CRC. (A) DEGs in six pairs CRC and PANT. (B) Representative immunofluorescence staining of TRPM7 (red) in CRC tissues compared with that observed in PANTs. Nuclei were stained using 4’,6-diamidino-2-phenylindole (blue). (C) The levels of TRPM7 mRNA in 15 CRC tissues and adjacent normal tissues were detected using qRT-PCR. The housekeeping gene GAPDH was used as an internal control. * $P<$ 0.01.

Figure 3.

TRPM7 was upregulated in CRC cells and TRPM7 deficiency inhibited the proliferation, migration and invasion of CRC cells. (A) The mRNA levels of TRPM7 in five human CRC cell lines and the human normal intestinal mucosa cell line NCM460 were detected using qRT-PCR. ** $P<$ 0.01. (B) qRT-PCR validated the efficiency of TRPM7 knockdown using siTRPM7 at the transcriptional level in CRC cells (HT29 and SW480). ** $P<$ 0.01 compared with that reported in cells treated with siCON. (C) The CCK-8 assay was used to assess the viability of CRC cells (HT29 and SW480) treated with siTRPM7 and siCON. ** $P<$ 0.01. (D) Representative images of the clonogenic survival assay in CRC cells (HT29 and SW480) treated with siTRPM7 and siCON. The number of colonies in each plate was analyzed statistically. * $P<$ 0.05. ** $P<$ 0.01. (E) The transwell assay showed that downregulated TRPM7 suppressed the migration of CRC cells. (F) The transwell assay showed that downregulated TRPM7 suppressed the invasion of CRC cells. The relative migration and invasion (number of cells) in each cell was analyzed statistically. ** $P<$ 0.01.

2.12 Statistical analysis

Statistical analyses were performed using R software (version 3.5.1, Vienna, Austria) [21, 22]. Data are shown as the mean $\pm$ standard deviation (SD). The Shapiro-Wilk test and Levene’s test were used to determine whether data were normally distributed and whether SD was homogeneous, respectively. Log2 transformations were performed for the qRT-PCR (2 ${}^{\rm-\Delta\Delta CT}$ ) data. For comparisons between two subgroups, differences in the means were examined using the paired $t$ -test. For comparisons between multiple groups, the means were compared using the Welch’s one-way ANOVA, followed by Tukey’s test for post-hoc pairwise comparisons. Additionally, Fisher’s exact probabilities and Spearman rank correlation were used to determine the correlation between TRPM7 expression and the clinicopathological characteristics of CRC patients. GraphPad Prism software (version 5.01, GraphPad, La Jolla, CA, USA) was used to create figures. A two-sided $P<$ 0.05 indicated statistical significance.

3. Results

3.1 Microarray datasets and differentially expressed genes

The GPL570 microarray platform was utilized for the mRNA expression profile datasets, including GSE41328, GSE39582, and GSE32323. In total, 44 CRC tissue samples and 44 PANT samples were analyzed. Using R software, 762, 1607, and 123 DEGs were identified among CRC tissues and PANTs from GSE41328, GSE39582, and GSE32323 datasets, respectively ( $P<$ 0.01, Fig. 1A). Moreover, out of all three datasets, 397 DEGs were found (Fig. 1B). Intriguingly, gene expression of TRPM7 was upregulated in CRC tissue among these DEGs, which is consistent with the outcomes of our microarray analysis (Fig. 2A). Therefore, we further assessed the gene functions of TRPM7 [5].

Table 2
Correlations between TRPM7 expression level and clinicopathological variables of 103 CRC patients

	Number	TRPM7 expression level		$P$ value
		Low	High
		( $n=$ 52)	( $n=$ 51)
Gender
Male	55	32	23	0.094
Female	48	20	28
Age, years
$<$ 60	51	27	24	0.622
$\geqslant$ 60	52	25	27
Tumor location
Colon	56	24	32	0.091
Rectum	47	28	19
Tumor size, cm
$<$ 5	41	25	16	0.083
$\geqslant$ 5	62	27	35
Serum CEA, ng/mL
$<$ 5	50	26	24	0.765
$\geqslant$ 5	53	26	27
Vascular invasion
Present	27	16	11	0.288
Absent	76	36	40
Lymphatic invasion
Present	28	13	15	0.615
Absent	75	39	36
Tumor infiltration
T1 and T2	32	23	9	0.004
T3 and T4	71	29	42
Lymph node metastasis
Present	40	12	28	0.001
Absent	63	40	23
Node counts
$<$ 12	32	15	17	0.623
$\geqslant$ 12	71	37	34
Distant metastasis
Present	14	3	11	0.019
Absent	89	49	40
Clinical stage
I	16	12	4	0.017
II	42	24	18
III	31	13	18
IV	14	3	11

Note: Bold italics indicate statistically significant values ( $P<$ 0.05).

3.2 TRPM7 overexpression in CRC tissues

In six pairs of specimens, microarray data from our medical center revealed the significant upregulation of TRPM7 gene expression in CRC tissues (Fig. 2A). Consistently, IF staining showed the upregulated expression of TRPM7 in CRC tissues, compared to the expression in PANTs ( $n=$ 15, Fig. 2B). These findings were corroborated by the qRT-PCR results ( $P<$ 0.01, $n=$ 15, Fig. 2C).

3.3 Associations of TRPM7 expression with clinicopathological parameters in CRC

The correlation between TRPM7 expression and the clinicopathological features was analyzed in 103 CRC patients. CRC patients were categorized into two groups: those with low TRPM7 expression ( $n=$ 52) and those with high TRPM7 expression ( $n=$ 51), in line with the median value of TRPM7 expression. The relationship between TRPM7 expression and the clinicopathological characteristics of CRC subjects is summarized in Table 2. Notably, significant correlations of high TRPM7 expression in CRC tissues were observed with tumor infiltration ( $P=$ 0.004), lymph node involvement ( $P=$ 0.001), clinical stage ( $P=$ 0.017), and distant metastasis ( $P=$ 0.019). Nevertheless, TRPM7 expression was not significantly associated with the remaining clinicopathological parameters, including age ( $P=$ 0.622), gender ( $P=$ 0.094), serum carcinoembryonic antigen (CEA, $P=$ 0.765), lymphatic invasion ( $P=$ 0.615), vascular invasion ( $P=$ 0.288), tumor size ( $P=$ 0.083), and tumor location ( $P=$ 0.091).

3.4 TRPM7 overexpression in CRC cell lines

Consistent with the results observed for the CRC tissue samples, mRNA expression of TRPM7 was significantly increased in the five CRC cell lines, especially in the HT29 and SW480 cells, in comparison with that in the human normal intestinal mucosa cell line NCM460 ( $P<$ 0.01, Fig. 3A).

Figure 4.

TRPM7 Knockdown triggered cell cycle arrest and promoted apoptosis in CRC cells. (A) Representative flow cytometry analysis of the cell cycle in CRC cells (HT29 and SW480) after transfection with siTRPM7 and siCON. Percentages (%) of cell populations at different stages of the cell cycle in HT29 and SW480 cells were statistically analyzed. * $P<$ 0.05, ** $P<$ 0.01, respectively. (B) Representative flow cyctometry analysis of cell apoptosis in CRC cells (HT29 and SW480) treated with siTRPM7 or siCON. The apoptotic rates (%) in HT29 and SW480 cells were analyzed statistically. * $P<$ 0.05.

Figure 5.

TRPM7 was related to the EMT pathway. (A) Representative immunofluorescence staining of TRPM7, E-cadherin, and N-cadherin (red) in CRC tissues compared with that observed in adjacent normal tissues. In CRC tissues, the expression of TRPM7 and N-cadherin was increased, whereas that of E-cadherin was reduced. Nuclei were stained using 4 ${}^{\prime}$ ,6-diamidino-2-phenylindole (blue). (B) Protein bands of TRPM7, E-cadherin, and N-cadherin in CRC tissues compared with those observed in PANTs. GAPDH was used as loading control. The relative expression levels of TRPM7, E-cadherin, and N-cadherin were statistically analyzed. ** $P<$ 0.01. (C) Protein bands of TRPM7, E-cadherin, and N-cadherin in CRC cells (HT29 and SW480). GAPDH was used as loading control. (D) Sestern blot showed that the protein level of E-cadherin was upregulated by treatment with siTRPM7 in CRC cells (HT29 and SW480). In contrast, the protein level of N-cadherin was downregulated.

3.5 Knockdown of TRPM7 significantly reduced the transcriptional and translational levels of TRPM7

siRNA transfection was performed to knockdown TRPM7 in HT29 and SW480 cells, to investigate the functions of TRPM7 in CRC cell lines. The efficiency of the knockdown was confirmed through qRT-PCR ( $P<$ 0.01, Fig. 3B). As a result, the mRNA expression of TRPM7 was drastically downregulated via TRPM7 siRNA in CRC cells.

3.6 Silencing of TRPM7 suppressed the proliferation of CRC cells

Results of the CCK-8 assay revealed that TRPM7 knockdown significantly suppressed the viability of CRC cells (HT29 and SW480) transfected with TRPM7-siRNA, in comparison with those transfected with siCON ( $P<$ 0.01, Fig. 3C). Additionally, the clonogenic survival assay indicated considerably decreased colony formation capacity in SW480 and HT29 cells treated with siTRPM7 compared to those transfected with siCON ( $P<$ 0.05, Fig. 3D). These results suggest that TRPM7 deficiency may hinder the proliferation of CRC cells.

3.7 Decreased TRPM7 inhibited migration and invasion of CRC cells

Results from the Transwell migration assay suggest that siTRPM7 transfection drastically reduced the migration of CRC cells ( $P<$ 0.01, Fig. 3E). Additionally, results from the Transwell invasion assay indicated that knockdown of TRPM7 in SW480 cells reduced cell invasion ( $P<$ 0.01, Fig. 3F).

3.8 Downregulation of TRPM7 triggered cell cycle arrest at the G0/G1 phase, reduced the number of the cells in the S phase, and promoted apoptosis

To identify the mechanism by which TRPM7 knockdown attenuated cell proliferation, flow cytometry was utilized for cell cycle and apoptosis analysis in HT29 and SW480 cells transfected with siTRPM7. Consequently, TRPM7 deficiency triggered significant cell cycle arrest at the G0/G1 phase and reduced the number of cells in the S phase in HT29 and SW480 cells compared to the control group ( $P<$ 0.05, Fig. 4A). Moreover, the downregulation of TRPM7 induced apoptosis in HT29 cells ( $P<$ 0.05, Fig. 4B).

3.9 TRPM7 deficiency reversed the dysregulation of EMT markers

Epithelial-mesenchymal transition has been shown to accelerate the metastasis of CRC cells. Therefore, both IF and western blot analysis were utilized to investigate EMT markers in CRC tissues and cells, including E-cadherin and N-cadherin. In contrast to the downregulation of E-cadherin and upregulation of N-cadherin in CRC tissues ( $P<$ 0.01, Fig. 5A and B), western blots also revealed that silencing of TRPM7 induced upregulated expression of E-cadherin and downregulated expression of N-cadherin in CRC cells ( $P<$ 0.05, Fig. 5C and D). These results suggest that the knockdown of TRPM7 may reduce the migration and invasion of CRC cells through the regulation of EMT.

4. Discussion

The TRPM7 gene has been implicated in numerous types of human malignancies [23], including esophageal [8], breast [9], gastric [10], bladder [11], and prostate cancers [12]. Moreover, a recent study conducted by Huang et al. [24] has revealed that the inhibition of TRPM7 suppressed CRC cell proliferation, implying that TRPM7 may serve as a cancer-promoting gene in CRC. However, there is no adequate evidence concerning the functional role of TRPM7 in CRC. Consequently, the present study was designed to explore the role of TRPM7 in CRC progression and the underlying mechanism. Our study revealed the significantly upregulated expression of the TRPM7 gene in CRC tissues in both the GEO datasets and our microarray data. Additionally, qRT-PCR data clearly demonstrated the significant upregulation of mRNA levels of TRPM7 in 15 CRC tissues in comparison with those of PANTs. These results were further validated by western blot analysis and IF staining. In exploration of the possible correlation between the mRNA expression of TRPM7 and clinicopathological features of CRC patients (Table 2), TRPM7 expression was positively related to tumor infiltration, clinical stage, lymph node involvement, and distant metastasis. However, TRPM7 expression was not significantly associated with gender, age, tumor location, tumor size, serum CEA, vascular invasion, or lymphatic invasion. Our results support the hypothesis that TRPM7 may serve as an oncogene in CRC.

Additionally, we further detected the mRNA levels of TRPM7 in different cell lines; these were higher in the five CRC cell lines than in the NCM460 cell line. In consideration of the dramatically upregulated TRPM7 expression in HT29 and SW480, the two cell lines were chosen for constructing TRPM7-deficient CRC cell lines using siRNA transfection. Consistent with the findings of a previous report [24], results from CCK-8 and clonogenic survival assays revealed that silencing of TRPM7 reduced the proliferation of CRC cells. Cell proliferation is largely affected by the cell cycle as well as apoptosis [25]. Thus, flow cytometry was used to analyze the cell cycle and apoptosis. As predicted, TRPM7 deficiency triggered significant cell cycle arrest at the G0/G1 phase and reduced the number of cells in the S phase in HT29 and SW480 cells. Moreover, the downregulation of TRPM7 induced the apoptosis of HT29 cells.

TRPM7 has been revealed to modulate the migration and invasion of human carcinoma cells [8, 9, 11, 12], indicating a potential role in cancer metastasis. Therefore, Transwell migration and invasion assays were performed to assess the impact of TRPM7 silencing on CRC cell migration and invasion. The results indicated that the knockdown of TRPM7 reduced cell migration and invasion in CRC cells. A plethora of studies has reported that EMT expedites cancer cell migration and invasion [12, 16, 17]. To unveil the mechanism underlying the regulatory role of TRPM7 in mediating CRC cell migration and invasion, IF staining and western blot analyses were performed to detect the expression of EMT markers, including E-cadherin and N-cadherin, in CRC tissues and cells (i.e., HT29 and SW480). In contrast to the downregulation of E-cadherin and upregulation of N-cadherin in CRC tissues, western blot analyses revealed that silencing of TRPM7 increased E-cadherin expression and reduced N-cadherin expression in CRC cells. These results suggested that TRPM7 deficiency may reverse the dysregulation of EMT markers. Further studies are warranted to address the limitations of the present study. For instance, investigation into the potential correlation between TRPM7 expression and the prognosis of CRC patients requires a longer follow-up time and a larger sample size. Moreover, pathways involved in the TRPM7 functional mechanism in CRC progression should also be elucidated in-depth.

5. Conclusion

In summary, our study revealed that high TRPM7 expression in CRC tissues is significantly related to deeper tumor infiltration, an advanced clinical stage, positive lymph node involvement, and distant metastasis. Moreover, silencing of TRPM7 in vitro suppressed CRC cell proliferation via the induction of cell cycle arrest at the G0/G1 phase, reduction in the number of cells in the S phase, and promotion of apoptosis. Furthermore, downregulation of TRPM7 in CRC cells inhibited cell migration and invasion by reversing EMT in vitro.

Footnotes

Acknowledgments

This work was supported by National Natural Science Foundation of China (81072152, 81770283), Natural Science Foundation of Hubei Province (2015CFA 027), Research Foundation of Health and Family Planning Commission of Hubei Province (WJ2015MA010, WJ2017M249), Clinical Medical Research Center of Peritoneal Cancer of Wuhan (2015060911020462) and Subsidy Project of No. 1 Hospital of Lanzhou University (ldyyyn2018-13).

Conflict of interest

The authors report no conflicts of interest in this work.

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TRPM7 deficiency suppresses cell proliferation,migration,and invasion in human colorectal cancer via regulation of epithelial-mesenchymal transition

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Microarray data processing

Table 1 List of primary antibodies

2.3 Cell culture

2.4 RNA isolation and RT-qPCR

2.5 siRNA transfection

2.6 Cell viability

2.7 Clonogenic survival

2.8 Flow cytometry

2.9 Transwell migration and invasion assays

2.11 Immunofluorescence staining

3. Results

3.1 Microarray datasets and differentially expressed genes

Table 2 Correlations between TRPM7 expression level and clinicopathological variables of 103 CRC patients

3.3 Associations of TRPM7 expression with clinicopathological parameters in CRC

3.4 TRPM7 overexpression in CRC cell lines

3.6 Silencing of TRPM7 suppressed the proliferation of CRC cells

3.7 Decreased TRPM7 inhibited migration and invasion of CRC cells

3.8 Downregulation of TRPM7 triggered cell cycle arrest at the G0/G1 phase, reduced the number of the cells in the S phase, and promoted apoptosis

3.9 TRPM7 deficiency reversed the dysregulation of EMT markers

4. Discussion

5. Conclusion

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
List of primary antibodies

Table 2
Correlations between TRPM7 expression level and clinicopathological variables of 103 CRC patients