MiR-200c-3p inhibits cell migration and invasion of clear cell renal cell carcinoma via regulating SLC6A1

Figures & data

Figure 1. SLC6A1 was high expressed in CCRCC tissues. (A) Abnormal expression mRNAs in CCRCC tissues were reflected by the volcano plot. (B) The heat map showed SLC6A1 was a higher-expression mRNA in CCRCC tissues than in adjacent tissues. (C) SLC6A1 was high expressed in tumor tissues according to qRT-PCR. *P<0.05, compared with adjacent tissues. (D) SLC6A1 was high expressed in tumor tissues according to western blot. *P<0.05, compared with adjacent tissues. (E) Patients with lower SLC6A1 expression had higher overall survival rate and longer survival time shown by Kaplan-Meier curve. (F) An ROC curve built on a univariate classification model based on SLC6A1 expression across independent TCGA dataset for predicting CCRCC.

Table 1. Clinicopathologic characteristics of patients in the study.

		The expression of SLC6A1
Clinicopathologic Characteristics		High expression(n = 56)	Low expression(n = 26)	P value
Gender	Male	42	19	0.8527
	Female	14	7
Age(years)	<60	26	14	0.5318
	≥60	30	12
Tumor size(cm)	<4	21	15	0.0864
	≥4	35	11
Clinical stage	I-II	8	10	0.0138
	III-IV	48	16
Tumor stage	T1-T2	31	8	0.0380
	T3-T4	25	18
Lymph node metastasis	Absence	36	7	0.0016
	Presence	20	19
Distant metastasis	Absence	34	9	0.0277
	Presence	22	17

* P < 0.5,

** P < 0.01 determined by Chi-Square test.

Table 2. Univariate and multivariate analyses of clinicopathological characteristics.

	Univariate analysis		Multivariate analysis
	HR (95% CI)	P	HR (95% CI)	P
laterality	1.454 ( 1.056 - 2.003 )	0.021	1.390 (0.962-2.009)	0.08
gender	0.950 (0.684-1.321)	0.762
age	0.823(0.458–1.478)	0.514
person neoplasm cancer status	6.080 ( 4.241 - 8.717 )	<0.001	3.540 ( 2.322 - 5.397 )	<0.001
lactate dehydrogenase	1.488 (0.507-4.363)	0.467
serum calcium	0.756 (0.530-1.079)	0.122
hemoglobin	2.109 ( 1.450 - 3.068 )	<0.001	1.364 (0.886-2.102)	0.159
platelet qualitative	2.415 ( 1.690 - 3.450 )	<0.001	1.680 ( 1.121 - 2.518 )	0.012
white cell count	0.696 (0.486-0.998)	0.047	0.998 (0.667-1.494)	0.992
stage event system version	0.698 (0.252-1.932)	0.486
stage event pathologic stage	4.591 ( 3.242 - 6.502 )	<0.001	2.406 ( 1.540 - 3.757 )	<0.001
Neoplasm histologic grade	2.838 ( 1.950 - 4.129 )	<0.001	1.168 (0.757-1.801)	0.484
T stage	5.149 ( 2.691 - 9.852 )	<0.001	1.975 (0.889-4.388)	0.095
N stage	1.559 (0.354-6.876)	0.555
M stage	0.253 (0.062-1.027)	0.038	0.367 (0.080-1.677)	0.196
Has-miR-200c	0.580 (0.344-0.977)	0.038	0.571 (0.325-1.002)	0.051
SLC6A1	1.498 ( 1.084 - 2.072 )	0.014	1.454 ( 1.056 - 2.003 )	0.021

^a The data were subjected to Cox's proportional hazards regression model. Bold italics indicate statistically significant values (P < 0.05).

^b Multivariate analysis used stepwise addition and removal of clinical covariates found to be associated with survival in univariate models (P < 0.05) and final models, include only those covariates that were significantly associated with survival (Wald statistic, P < 0.05). Bold italics indicate statistically significant values (P < 0.05).

Figure 2 . SLC6A1 knockdown inhibited CCRCC cells to proliferate, migrate and invade. (A) The transfection efficiency was observed by RT-qPCR. *P < 0.05, compared with control group. (B) SLC6A1 siRNA transfection inhibited the proliferation of 786-O cells. *P<0.05, compared with control group. (C-D) SLC6A1 siRNA transfection inhibited the migration and invasion of 786-O cells (200 ×). *P < 0.05, compared with control group.

Figure 3 . MiR-200c-3p directly targeted at SLC6A1. (A) MiR-200c-3p was predicted to target SLC6A1 and the targeting scheme was illustrated. (B) MiR-200c-3p could directly targeted at SLC6A1 3’UTR. *P < 0.05, compared with NC mimics group. (C) MiR-200c-3p was low expressed in tumor tissues by qRT-PCR detection. *P < 0.05, compared with adjacent tissues. (D) MiR-200c-3p was negatively related with SLC6A1 expression with P < 0.01 and R² = 0.524.

Figure 4 . MiR-200c-3p reversed the progression of SLC6A1. (A) MiR-200c-3p mimics transfection downregulated SLC6A1 expression while SLC6A1 cDNA upregulated SLC6A1 expression. *P < 0.05, compared with control group. (B) MiR-200c-3p mimics transfection decreased proliferation but SLC6A1 cDNA transfection increased proliferation in 786-O cells. Meanwhile, there was no significant difference between miR-200c-3p+SLC6A1 group and control group. *P < 0.05, compared with control group. (C-D) MiR-200c-3p mimics transfection inhibited but SLC6A1 cDNA transfection increased the migration and invasion in 786-O cells. There was no significant difference between miR-200c-3p+SLC6A1 group and control group despite of migration or invasion (200 ×). *P < 0.05, compared with control group.

Figure 5 . Effects of the miR-200c-3p and SLC6A1 on CCRCC in vivo. (A) Tumor specimens from nude mice divided into five groups. (B) Tumor weights from nude mice divided into five groups. *P < 0.05, compared with control group. (C) The number of murine pulmonary nodules determined by dissecting microscope. *P < 0.05, compared with control group. (D) The weight of lungs in nude mice from 5 respective groups. *P < 0.05, compared with control group. (E) H&E staining was conducted to detect the number of lung metastatic nodules (× 40). *P < 0.05, compared with control group.

Table 3. Primer sequences used in qRT-PCR.

			Primers
miR-200c		F	5’- TCGTCTTACCCAGCAGTG-3’
		R	5’- CGGCAGTATTAGAGACTCC-3’
U6		F	5’-CTCGCTTCGGCAGCACATA-3’
		R	5’-AACGATTCACGAATTTGCGT-3’
SLC6A1		F	5’-GTGAGGCAACTCCAAGGT-3’
		R	5’-CAGATGGACGTGCGATGT-3’
GAPDH		F	5’-GAAGGTGAAGGTCGGAGTC-3’
		R	5’-GAAGATGGTGATGGGATTTC-3’

F: forward primers; R: reverse primers.