Supplementary Materials microrna-200b and microrna-200c promote colorectal cancer cell proliferation via targeting the reversion-inducing cysteine-rich protein with Kazal motifs Supplementary Table 1. Patients Characteristics. Case No. Clinical History Gender Age (years) TNM Stage CRC#1 colon carcinoma male 79 Ⅱ CRC#2 colon carcinoma male 85 Ⅱ CRC#3 colon carcinoma Female 63 Ⅱ CRC#4 colon carcinoma Female 51 Ⅱ CRC#5 colon carcinoma male 69 Ⅱ CRC#6 colon carcinoma Female 75 Ⅱ CRC#7 colon carcinoma male 61 Ⅱ CRC#8 colon carcinoma male 83 Ⅱ CRC#9 colon carcinoma male 62 Ⅱ CRC#10 colon carcinoma male 58 Ⅱ CRC#11 colon carcinoma Female 58 Ⅱ CRC#12 colon carcinoma male 66 Ⅱ CRC#13 colon carcinoma Female 69 Ⅱ CRC#14 colon carcinoma Female 44 Ⅱ CRC#15 colon carcinoma male 56 Ⅱ 1
Supplementary Figure Legends Supplementary Figure 1. Comparison of mir-200b/c and RECK levels in different colorectal cancer cells. (A) Comparison of mir-200b/c levels in HT29, Caco-2 and SW480 cells. (B) Comparison of RECK protein levels in HT29, Caco-2 and SW480 cells. Left panel: representative image; right panel: quantitative analysis (**P < 0.01).. Supplementary Figure 2. Overexpression of mir-200b/c and regulation of RECK expression by mir-200b/c in Caco-2, HT29 and SW480 cells. (A and B) Quantitative RT-PCR analysis of mir-200b and mir-200c levels in Caco-2 cells treated with pre-scramble, pre-mir-200b, pre-mir-200c, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) and in cells treated with anti-scramble, anti-mir-200b, anti-mir-200c, or anti-mir-200b plus anti-mir-200c (in a 0.5:0.5 ratio) (**P < 0.01). (C and D) Quantitative RT-PCR analysis of mir-200b and mir-200c levels in HT29 cells treated with pre-scramble, pre-mir-200b, pre-mir-200c, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) and in cells treated with anti-scramble, anti-mir-200b, anti-mir-200c, or anti-mir-200b plus anti-mir-200c (in a 0.5:0.5 ratio) (**P < 0.01). (E and F) Quantitative RT-PCR analysis of mir-200b and mir-200c levels in SW480 cells treated with pre-scramble, pre-mir-200b, pre-mir-200c, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) and in cells treated with anti-scramble, anti-mir-200b, anti-mir-200c, or anti-mir-200b plus anti-mir-200c (in a 0.5:0.5 ratio) (**P < 0.01). (G) Quantitative RT-PCR analysis of mir-200b and mir-200c levels in Caco-2 cells infected with control lentivirus or lentivirus to overexpress mir-200b, mir-200c, or mir-200b plus mir-200c (**P < 0.01).(H and I) Western blotting analysis of RECK protein levels in HT29 cells treated with pre-scramble, pre-mir-200b, pre-mir-200c, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) and in cells treated with anti-scramble, anti-mir-200b, anti-mir-200c, or anti-mir-200b plus anti-mir-200c (in a 0.5:0.5 ratio). Left panel: representative image; right panel: quantitative analysis (**P < 0.01). (J) Quantitative RT-PCR analysis of RECK mrna levels in HT29 cells treated with pre-scramble, pre-mir-200b, pre-mir-200c, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) and in cells treated with anti-scramble, anti-mir-200b, anti-mir-200c, or anti-mir-200b plus anti-mir-200c (in a 0.5:0.5 ratio). (K and L) Western blotting analysis of RECK protein levels in SW480 cells 2
treated with pre-scramble, pre-mir-200b, pre-mir-200c, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) and in cells treated with anti-scramble, anti-mir-200b, anti-mir-200c, or anti-mir-200b plus anti-mir-200c (in a 0.5:0.5 ratio). Left panel: representative image; right panel: quantitative analysis (**P < 0.01). (M) Quantitative RT-PCR analysis of RECK mrna levels in SW480 cells treated with pre-scramble, pre-mir-200b, pre-mir-200c, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) and in cells treated with anti-scramble, anti-mir-200b, anti-mir-200c, or anti-mir-200b plus anti-mir-200c (in a 0.5:0.5 ratio). Supplementary Figure 3. Selection of efficient sirna against RECK and Downregulation of RECK expression by sirna and upregulation of RECK expression by overexpression vector in colorectal cancer cells. (A) Three sirna sequences targeting different sites of human RECK cdna were designed. (B and C) Western blotting analysis of RECK protein levels in Caco-2 cells treated with different scrambled control sirnas and different RECK sirnas. B: representative image; C: quantitative analysis (**P < 0.01). The sequence with the best interfering effect (named RECK sirna1) was selected and used in further studies.(d) Western blotting analysis of protein levels of RECK, SKP2, p27 Kip1 in Caco-2cells treated with scrambled control sirna, RECK sirna, control vector, or RECK vector. Left panel: representative image; right panel: quantitative analysis (**P < 0.01). (E) Western blotting analysis of RECK protein levels in HT29 cells treated with scrambled control sirna, RECK sirna, control vector, or RECK vector. Left panel: representative image; right panel: quantitative analysis (**P < 0.01). (F) Western blotting analysis of RECK protein levels in SW480 cells treated with scrambled control sirna, RECK sirna, control vector, or RECK vector. Left panel: representative image; right panel: quantitative analysis (**P < 0.01). Supplementary Figure 4. MTT assay analysis and EdU proliferation assay analysis of the effect of RECK-targeted mir-200b/c on the growth of colorectal cancer cells. (A and B) The MTT viability assay was performed 12, 24, 36, 48, 60 and 72 h after the transfection of HT29 cells with scrambled negative control RNA, pre-mir-200b/c, or anti-mir-200b/c. (C) The MTT viability assay was performed 12, 24, 36, 48, 60 and 72 h after the transfection of HT29 cells with scrambled control sirna, RECK sirna, control vector or the RECK overexpression 3
vector. (D and E) The MTT viability assay was performed 12, 24, 36, 48, 60 and 72 h after the transfection of SW480 cells with scrambled negative control RNA, pre-mir-200b/c, or anti-mir-200b/c. (F) The MTT viability assay was performed 12, 24, 36, 48, 60 and 72 h after the transfection of SW480 cells with scrambled control sirna, RECK sirna, control vector or the RECK overexpression vector. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (G and H) The EdU proliferation assay was performed 48 h after the transfection of HT29 cells with scrambled negative control RNA, pre-mir-200b/c, or anti-mir-200b/c. Left panel: representative image; right panel: ratio of EdU-positive HT29 cells (**P < 0.01). (I) The EdU proliferation assay was performed 48 h after the transfection of HT29 cells with scrambled control sirna, RECK sirna, control vector or the RECK overexpression vector. Left panel: representative image; right panel: ratio of EdU-positive HT29 cells (**P < 0.01). (J and K) The EdU proliferation assay was performed 48 h after the transfection of SW480 cells with scrambled negative control RNA, pre-mir-200b/c, or anti-mir-200b/c. Left panel: representative image; right panel: ratio of EdU-positive SW480 cells (**P < 0.01). (L) The EdU proliferation assay was performed 48 h after the transfection of SW480 cells with scrambled control sirna, RECK sirna, control vector or the RECK overexpression vector. Left panel: representative image; right panel: ratio of EdU-positive SW480 cells (**P < 0.01). Supplementary Figure 5. The effect of mir-200b/c and RECK on the migration of colorectal cancer cells. (A) Analysis of the migration ability of Caco-2 cells transfected with pre-scramble, pre-mir-200b, pre-mir-200c, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) using wound healing assay. (B) Analysis of the migration ability of Caco-2 cells transfected with anti-scramble, anti-mir-200b, anti-mir-200c, or anti-mir-200b plus anti-mir-200c (in a 0.5:0.5 ratio) using wound healing assay. (C) Analysis of the migration ability of Caco-2 cells transfected with scrambled control sirna, RECK sirna, control vector, or RECK vector using wound healing assay. Left panel: representative image; right panel: quantitative analysis. (**P < 0.01). Supplementary Figure 6. The effects of mir-21 on RECK in combination with mir-200b/c. 4
(A) Quantitative RT-PCR analysis of mir-21, mir-200b and mir-200c levels in Caco-2 cells treated with pre-scramble, pre-mir-21, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio) (**P < 0.01). (B) Quantitative RT-PCR analysis of RECK mrna levels in Caco-2 cells treated with pre-scramble, pre-mir-21, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio). (C) Western blotting analysis of RECK protein levels in Caco-2 cells treated with pre-scramble, pre-mir-21, or pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio). Left panel: representative image; right panel: quantitative analysis (**P < 0.01). (D) The MTT viability assay was performed 12, 24, 36, 48, 60 and 72 h after the transfection of Caco-2 cells with pre-scramble, pre-mir-21, pre-mir-200b plus pre-mir-200c (in a 0.5:0.5 ratio), or pre-mir-21 plus pre-mir-200b plus pre-mir-200c (in a 0.5:0.25:0.25 ratio) (*P < 0.05; **P < 0.01). (E) Quantitative RT-PCR analysis of the expression levels of mir-21 (in the form of mirna/u6 ratio) in eight pairs of colorectal cancer (CRC) and normal adjacent tissue (NAT) samples (*P < 0.05; **P < 0.01). 5
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