promote ROS production and metastasis of HCC cells

Transcription

1 MCU-dependent mitochondrial Ca 2+ inhibits NAD+/SIRT3/SOD2 pathway to promote ROS production and metastasis of HCC cells Tingting Ren 1#, Hui Zhang 2#, Jiaojiao Wang 1, Jianjun Zhu 1, Mingpeng Jin 1,Yousheng Wu 1, Xu Guo 1, Lele Ji 1, Qichao Huang 1, Hongxin Zhang 2, Hushan Yang 3, Jinliang Xing 1* 1

2 SUPPLEMENTAL TABLE Table S1. Primary antibodies used for immunohistochemistry and western blot. Antibody Company (Cat. No.) Working dilutions MCU SIGMA(HPA016480) WB: 1/250 IHC:1/100 MICU1 NOVUS (NBP ) WB: 1/1000 IHC:1/100 MICU2 Abcam (ab101465) WB: 1/500 IHC:1/100 β actin Beijing TDY BIOTEC (TDY051C) WB: 1/3000 JNK Proteintech ( AP) WB: 1/1000 p-jnk affinity (AF3319) WB: 1/500 IHC:1/100 p38 Proteintech ( AP) WB: 1/1000 p-p38 affinity (AF4001) WB: 1/500 ERK Proteintech ( AP) WB: 1/1000 p-erk Cell Signaling (#4370) WB: 1/1000 paxillin affinity (AF6331) WB: 1/500 p-paxillin Abcam (ab193677) WB: 1/500 SOD2 Proteintech ( AP) WB: 1/500 IP:1/250 Acetyl-lysine Cell Signaling (#9441) WB: 1/1000 SIRT3 Cell Signaling (#5490) WB: 1/1000 MMP2 Proteintech ( AP) WB: 1/500 MMP9 Proteintech ( AP) WB: 1/500 Vinculin Abcam(ab18058) IF: 1/200 PDH Santa Cruz Biotechnology (sc ) WB: 1/1000 2

3 p-pdh Millipore (ABS204) WB: 1/2000 NCLX SIGMA(HPA040668) WB: 1/200 IP:1/25 SERCA3 Abcam(ab54876) WB: 1/1000 IP3R2 Abcam(ab104550) WB: 1/500 IP3R3 Abcam(ab55983) WB: 1/500 Parvalbumin SIGMA(P 3088) WB: 1/1000 3

4 Table S2. Distribution of HCC patients' characteristics. Variable All patients, n (%) n = 201 MCU Expression P value MICU1 Expression P value low high low high Gender, n (%) Female 27 (13.4) Male 174 (86.6) Age, years < (51.7) (48.3) HBsAg, n (%) Negative 14 (7.0) Positive 187 (93) Serum AFP, n (%), ng/ml < (54.7) (45.3) Differentiation, n (%) I + II 66 (32.8)

5 III + IV 135 (67.2) TNM stage, n (%) I + II 142 (70.6) III + IV 59 (29.4) Treatment, n (%) Surgery 89 (44.3) Surgery+TACE 112 (55.7) Death, n (%) Yes 83 (41.3) No 118 (58.7) Recurrence, n (%) Yes 116 (57.7) No 85 (42.3)

6 Table S3. Public datasets used for bioinformatic analysis GEO Accession No. Platform Probes /Genes HCC Sample No. Patient Ethnicity Etiology Source URL GSE25097 Rosetta/Me rck Human RSTA Affymetrix / Chinese HBV m.nih.gov/geo/quer y/acc.cgi?acc=gse Rosetta/Me GSE22058 rck Human RSTA 43483/ Chinese HBV m.nih.gov/geo/quer y/acc.cgi?acc=gse Custom Affymetrix Human Genome U133A / GSE14520 Array Affymetrix / HBV m.nih.gov/geo/quer y/acc.cgi?acc=gse HT Human Genome U133A Array TCGA Illumina Hiseq/GA / 201 / HBV/ HCV 6

7 Table S4. Sequence of primers 1. Primers used in q-pcr analysis MCU MICU1 MICU2 MCUb EMRE GAPDH forward primer reverse primer forward primer reverse primer forward primer reverse primer forward primer reverse primer forward primer reverse primer forward primer reverse primer GCAGAATTTGGGAGCTGTTT GTCAATTCCCCGATCCTCTT GAGGCAGCTCAAGAAGCACT CAAACACCACATCACACACG GGCAGTTTTACAGTCTCCGC AAGAGGAAGTCTCGTGGTGTC CCCCAGGTTTTGCGTGTGA GTGGCACCACGGTACTATAATG TGTCGGGACACTCATTAGCA GCTGATAGGGAAGGCAGAGA GGAGCGAGATCCCTCCAAAAT GGCTGTTGTCATACTTCTCATGG 2. Primers used in gene cloning MCU Parvalbumin (PV) PV-Mito SIRT3 SOD2 3. sirna MCU forward primer reverse primer forward primer reverse primer forward primer reverse primer forward primer reverse primer forward primer reverse primer sense antisense GCGGATCCCGTTTCCAGTTGAGAGATGGCGGCC GCGAATTCGCCAGGATTCAGAGGCTTTTTGCAG TATAAGCTTATGACAGACTTGCTGAACGCTGAGGACATC GCTGGATCCGCTTTCAGCCACCAGAGTGGAGAATTCGTC ACAGATCTGCCACCATGTCCGTCCTGACGCCG AGACTCGAGCGGCCGCTTTAGCTTTCAGCCACCAGAGTGG CGGGATCCAACATGGCGTTCTGGGGTTG CCGCTCGAGGGCAGCCATCATCCTATTTG CGGAATTCTAGCAGCATGTTGAGCCGGG CGGAATTCAGATCGGCGGCATCAGCGGTAG CCUAGAGAAAUACAAUCAACUCAdAdG CUUGAGUUGAUUGUAUUUCUCUAGGUC SIRT3 sense GCTTGATGGACCAGACAAA 7

8 SOD2 Paxillin antisense sense antisense sense antisense TTTGTCTGGTCCATCAAGC CCCTGGAACCTCACATCAA TTGATGAGAGGTTCCAGGG CCCUGACGAAAGAGAAGCCUAUU UAGGCUUCUCUUUCGUCAGGGUU 8

9 Supplemental materials and methods Collection of tissue samples and clinical data Tissue samples from 201 HCC patients were collected at Xijing Hospital affiliated with the Fourth Military Medical University in Xi an, China. The eligibility criteria for HCC patient recruitment were set as follows: (1) histologically-confirmed hepatocellular carcinoma (HCC); (2) receiving surgical resection; (3) availability of complete clinical and follow-up data; (4) no preoperative anticancer treatment; (5) no history of other malignancy; and (6) alive at least 1 month after surgery. The last follow-up date was February 2016 and the median follow-up duration was 28.5 months (ranging from 2.4 to 85.6 months). The study was approved by the Ethics Committee of the Fourth Military Medical University and written informed consent was obtained from all participants. 9

10 SUPPLEMENTAL FIGURES LEGENDS Figure S1 related to Figure 1. (A-E) The relative mrna expression levels of tumor and peritumor of MCU, MICU1, MICU2, MCUb and EMRE were analyzed in public microarray data GSE22058, GSE25097, GSE14520 and TCGA downloaded from the Gene Expression Omnibus (GEO) database (no EMRE data in GSE14520). (F and G) Representative IHC staining images of MCU, MICU1 and NCLX in pairedtumor and peritumor HCC tissues (n = 201). (H) Representative immunohistochemical staining images of MCU and MICU1 in primary and metastatic HCC tissues (n = 60). (I) Representative IHC staining images of MCU and MICU1 in normal and fatty human liver tissues (n = 10). (J) Representative IHC staining images of MCU and MICU1 in liver tissues of control and ob/ob mice (n = 6). Scale bar: 100 μm. Figure S2 related to Figure 2. (A) and (B) Western blot analysis of MCU and MICU1 protein levels in HCC cell lines and stably transfected HCC cells. (C) Co-localization of the mitochondria labeled with Mito-tracker (Red) and mitochondrial Ca 2+ detected by mitopericam (Green) in SMMC7721 and MHCC97H cells. Scale bar: 20 μm. (D) Representative confocal microscope images of mitochondrial Ca 2+ levels ([Ca 2+ ] m ) detected by mitopericam in normal hepatocyte HL-7702, HCC cells with abilities of high invasiveness and low invasiveness. Scale bar: 20 μm. (E) Representative confocal microscope images of mitochondrial Ca 2+ levels ([Ca 2+ ] m ) detected by mitopericam 10

11 in stably transfected HCC cells. Scale bar: 20 μm. (F) Representative time-course recording of [Ca 2+ ] m fluorescence detected by mitopericam. After 20 s baseline recording, [Ca 2+ ] m responses to 10 μm histamine in stably transfected HCC cells was investigated. Ca 2+ response signals were presented as maximal amplitude fluorescence intensity. (G) Western blot analysis of MICU1 and MICU2 protein levels. (H) Representative traces and quantification of [Ca 2+ ] m in HCC cells. (I) Western blot analysis of SERCA3, IP3R2 and IP3R3 protein levels in HCC cells as indicated. * P < 0.05; ** P < Figure S3 related to Figure 3. (A) Intracellular ROS levels were analyzed by flow cytometry after staining with specific fluorescence dye DCFH-DA in HCC cells as indicated. (B) Co-localization of the mitochondria labeled with Mito-tracker Red (Red) and PV-Mito-GFP (Green) in MHCC97H cells. And representative time-course recording of [Ca 2+ ] m fluorescence detected by mitopericam. After 20 s baseline recording, [Ca 2+ ] m responses to 10 μm histamine in MHCC97H cells was investigated. Ca 2+ response signals were presented as maximal amplitude fluorescence intensity. (C) Cell proliferation ability was evaluated using EdU incorporation assay and quantified by the percentage of EdU-positive cells (red) in SMMC7721 and MHCC97H cells. Hoechst was used to label cell nuclei (blue). (D, F) Transwell assay for migration (upper) and invasion (down) ability of HCC cells. (E, G) Wound healing assays for migration rate in HCC cells. (H) MitoTEMPO (5 μm) was used to 11

12 scavenge mitochondrial ROS and mitochondrial ROS was assessed by staining with MitoSOX red (4 μm) for 10 minutes. Representative confocal microscope images (left) and quantification of fluorescence (right) were shown. Figure S4 related to Figure 4. (A and B) Protein expression levels of PDH, p-pdh, SIRT3 and SOD2 in HCC stably transfected cells. (C) Mitochondrial NAD + /NADH ratio were determined in HCC cells. FK866 (10 μm, for 72 hours), NAM (1 mm, for 72 hours). (D) Western blot analysis of SIRT3 protein expression level in HCC cells. (E) SIRT3 enzyme activity was determined in HCC cells. Figure S5 related to Figure 5. (A) Western blot analysis of SOD2 expression level in HCC cells which are transiently transfected with sisod2 or SOD2 expression vector for 48 hour. SOD2: SOD2 expression vector; sisod2: sirna against SOD2. (B) SOD2 enzyme activity was determined in HCC cells. (C) Expression and acetylation level of SOD2 in HCC cells. Figure S6 related to Figure 6. (A) Western blot analysis of p-jnk, JNK, Paxillin and p-paxillin levels in MHCC97H cells. (B) The correlation between the protein expression levels of MCU and p-jnk 12

13 in 201 HCC tissues. Representative IHC staining images of MCU and p-jnk in HCC tissues. (C) Western blot analysis of HIF1-α levels in HCC cells. Figure S7 related to Figure 7. Transwell assay for migration (A) and invasion (B) ability of HCC cells. (C) Wound healing assays for migration rate in HCC cells. Figure S8 related to Figure 8. (A) Representative IHC staining images of PV in tumor and peritumor from HCC xenograft nude mice model. 13

14 FigureS1 A B C D E

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16 FigureS2 A B C D E

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