Supplementary Materials For

microRNA-133a, downregulated in osteosarcoma, suppresses

proliferation and promotes apoptosis by targeting Bcl-xL and Mcl-1

Fang Ji1, Hao Zhang1, Yang Wang1, Ming Li, Weidong Xu, Yifan Kang,

Zhiwei Wang, Zimin Wang, Ping Cheng, Dake Tong, Cheng Li, Hao

Tang*

Department of Orthopedics, Changhai Hospital, Second Military Medical University,

Shanghai 200433, China

* Corresponding author Address: Department of Orthopedics, Changhai Hospital,

Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.

Tel.: (+86-21) 31161682; Fax: (+86-21) 81871398.

E-mail: tanghao1978@163.com

1 Equal contribution.

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Supplementary Table 1. Characteristics of osteosarcoma patients

No. Sex Age Diagnosis LocusClinicalStages

1 F 18 Primary OS femur Ⅰ2 M 33 Primary OS femur ⅡA3 M 16 Primary OS femur III4 M 17 Primary OS femur ⅡB5 M 30 Primary OS femur ⅡB6 M 24 Primary OS femur III7 F 18 Primary OS tibia ⅡB8 M 57 Primary OS femur Ⅰ9 M 24 Primary OS femur ⅡB10 F 17 Primary OS femur Ⅲ11 M 20 Primary OS femur ⅡA12 M 21 Primary OS femur Ⅰ13 M 8 Primary OS tibia Ⅲ14 M 19 Primary OS femur ⅡB15 M 48 Primary OS femur ⅡA16 M 13 Primary OS fibula ⅡA17 M 35 Primary OS femur Ⅰ18 M 16 Primary OS costa ⅡA19 F 21 Primary OS femur Ⅰ20 M 11 Primary OS humerus ⅡB21 F 17 Primary OS femur ⅡA22 M 47 Primary OS femur ⅡA23 M 59 Primary OS femur Ⅰ24 M 32 Primary OS femur ⅡA25 M 21 Primary OS tibia ⅡA26 F 42 Primary OS humerus ⅡA27 F 18 Primary OS tibia ⅡA28 M 20 Primary OS tibia ⅡA29 F 23 Primary OS femur Ⅲ30 M 16 Primary OS femur ⅡB31 F 46 Primary OS radix Ⅰ32 F 19 Primary OS femur ⅡA33 F 41 Primary OS femur ⅡA34 M 33 Primary OS tibia Ⅰ35 M 37 Primary OS tibia ⅡB36 M 16 Primary OS humerus ⅡA37 M 13 Primary OS tibia ⅡA

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38 M 8 Primary OS femur ⅡA39 M 23 Primary OS tibia ⅡB40 M 31 Primary OS femur ⅡB41 M 23 Primary OS femur Ⅲ42 M 26 Primary OS mandible ⅡA43 M 19 Primary OS fibula ⅡA44 M 23 Primary OS femur ⅡA45 F 17 Primary OS femur Ⅲ46 F 38 Primary OS fibula ⅡA47 M 45 Primary OS costa ⅡA48 M 14 Primary OS fibula Ⅲ49 F 16 Primary OS tibia Ⅰ50 M 17 Primary OS pubis Ⅲ51 F 10 Primary OS femur Ⅲ52 M 16 Primary OS femur ⅡB53 M 17 Primary OS ilium ⅡB54 M 16 Primary OS femur ⅡB55 M 11 Primary OS tibia Ⅰ56 M 18 Primary OS tibia ⅡB57 M 20 Primary OS femur ⅡB58 F 69 Primary OS vertebrate ⅡB59 M 40 Primary OS pelvis ⅡB61 M 26 Primary OS femur I60 M 10 Primary OS femur Ⅲ62 M 73 Primary OS humerus Ⅲ63 M 18 Primary OS tibia ⅡB64 M 34 Primary OS pelvis ⅡB65 M 47 Primary OS ilium Ⅰ66 M 49 Primary OS femur ⅡB67 M 35 Primary OS femur Ⅲ68 M 13 Primary OS femur ⅡA69 M 55 Primary OS vertebrate ⅡB70 M 15 Primary OS femur ⅡB71 F 19 Primary OS clavicle ⅡA72 F 12 Primary OS femur ⅡA73 M 51 Primary OS radius ⅡB74 M 7 Primary OS ilium ⅡB75 F 16 Primary OS femur ⅡA76 F 26 Primary OS femur ⅡB77 F 20 Primary OS femur ⅡB78 F 17 Primary OS femur ⅡA79 M 36 Primary OS femur ⅡB80 M 19 Primary OS femur ⅡB

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81 M 18 Primary OS femur ⅡB82 F 37 Primary OS tibia ⅡB83 M 14 Primary OS tibia ⅡB84 M 54 Primary OS femur ⅡB85 M 20 Primary OS pubis ⅡB86 F 61 Primary OS femur ⅡB87 M 15 Primary OS femur Ⅲ88 F 25 Primary OS femur ⅡB89 F 22 Primary OS fibula ⅡB90 M 24 Primary OS femur ⅡB91 F 10 Primary OS radius ⅡB92 F 14 Primary OS femur ⅡB

Abbreviations: M, male; F, female; OS, osteosarcoma.

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Supplementary Table 2. Enrichment analysis of predicted miR-133a targets in KEGG cell signaling pathway database.

Pathway Count p value

Pathways in cancer 21 0.001

PI3K-Akt signaling pathway 20 0.006

MAPK signaling pathway 16 0.010

Endocytosis 16 0.014

Transcriptional misregulation in cancer 14 0.046

Wnt signaling pathway 13 0.047

Regulation of actin cytoskeleton 10 0.052

HTLV-I infection 10 0.098

Morphine addiction 10 0.104

Oocyte meiosis 10 0.130

Only the most enriched 10 cell signaling pathways of predicted miR-133a targets in

KEGG database were shown. A hypergeometric test was used to validate the

significance with p values shown as indicated.

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Supplementary Fig . 1. Transfection of miR-133a mimics restored its expression in

osteosarcoma cells. (A) Osteosarcoma MG63 and U2OS cells were transfected with

Ctrl RNA or miR-133a mimics as indicated. miR-133a expression in the transfected

cells was detected by qRT-PCR. (B) Shown is qRT-PCR detection of miR-133a

expression in the transfected osteosarcoma cells during the indicated time periods

after inoculation in nude mice. Data are shown as mean ± s.d. (n = 4 ) . S imilar results

were obtained in three independent experiments. ** , p <0.01.

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Supplementary Fig . 2 . miR- 133a inhibition enhances cell proliferation, inhibits cell

apoptosis , and upregulates Bcl-xL and Mcl-1 expression in hFOB 1.19 cells. (A)

Human normal osteoblast hFOB 1.19 cells were transfected with Ctrl inhibitor or

miR-133a inhibitor as indicated. miR-133a expression in the transfected cells were

detected by qRT-PCR. (B) hFOB 1.19 cells were transfected with Ctrl inhibitor or

miR- 133a inhibitor as indicated. Cell proliferation was measured in the indicated time

periods post transfection using MTT assay. ( C ) hFOB 1.19 cells were transfected with

Ctrl inhibitor or miR- 133a inhibitor as indicated and treated with serum deprivation

and hypoxia for 48 hrs. Cells were stained with PI and Annexin V, and analyzed using

FACS. (D) hFOB 1.19 cells were transfected with Ctrl inhibitor or miR- 133a inhibitor

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as indicated . Expression of Bcl-xL, Mcl-1 , and internal control β-actin w as detected

by Western blot. Data are shown as mean ± s.d. (n = 4 ) or presented separately of one

representative experiment . S imilar results were obtained in three independent

experiments. * , p <0.0 5; ** , p <0.01.

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Supplementary Figure 3. Enrichment analysis of predicted miR-133a targets in

KEGG cell signaling pathway database

Shown is KEGG pathway in cancer, the most significantly enriched by the predicted

targets of miR-133a (p=0.001). Red frame shows the predicted miR-133a targets.

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