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Prognostic Value of TIMM50 Expression in Colorectal Cancer

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Prognostic Value of TIMM50 Expression in Colorectal Cancer Clinical research Prognostic value of TIMM50 expression in colorectal cancer Bo Sun1, Jun Wang2, Yan-Feng Zhu3, Zhen-Yang Li2, Jian-Bin Xiang2, Zong-You Chen2, Zhi-Gang He4, Xiao-Dong Gu2 1Department of Gastric Surgery, Fudan University Shanghai Cancer Center, Shanghai, Corresponding authors: China 200032 Zhi-Gang He 2Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, Department of General China 200040 Surgery 3Department of Nursing, Huashan Hospital, Fudan University, Shanghai, China 200040 Shanghai Songjiang District 4Department of General Surgery, Shanghai Songjiang District Central Hospital, Central Hospital Shanghai, China 201600 746 Zhongshan Middle Road Shanghai, China 201600 Submitted: 8 April 2019 Phone: +86-21-67720001 Accepted: 12 September 2019 E-mail: [email protected] Arch Med Sci Xiao-Dong Gu DOI: https://doi.org/10.5114/aoms.2020.94487 Department of General Surgery Copyright © 2020 Termedia & Banach Huashan Hospital Fudan University 12 Wulumuqi Middle Road Abstract Shanghai, China 200040 Phone: +86-21-52887333 Translocase of the inner mitochondrial membrane 50 (TIMM50) Introduction: E-mail: [email protected] is universally considered to play a key role in several malignancies. Howev- er, its role in predicting colorectal cancer (CRC) patient prognosis remains unclear. Material and methods: A total of 192 CRC patients (123 men and 69 women) who underwent radical resection participated in this study. The patients were followed up every three months after surgery for five years. TIMM50 expression in tumour tissues was measured by quantitative real-time PCR, Western blotting and immunohistochemistry. TIMM50 expression was stud- ied to assess correlations with clinicopathological factors and survival time. Results: TIMM50 expression increased significantly in CRC tumour tis- sues. Moreover, high TIMM50 expression was related to pathologic stage (p = 0.043), N stage (p = 0.048) and distant metastasis (p = 0.015), but TIMM50 expression was not related to other clinical factors. A Kaplan-Meier survival analysis indicated that patients with low TIMM50 expression had a longer overall survival than those with high TIMM50 expression (p = 0.002). Fur- thermore, distant metastasis and high TIMM50 expression were confirmed as independent prognostic factors for the overall survival of CRC patients in a multivariate analysis (p = 0.003). Conclusions: TIMM50 may be a key factor for monitoring CRC and a new prog- nosis indicator for CRC patients. Key words: colorectal cancer, translocase of the inner mitochondrial membrane 50, prognosis, survival. Introduction Colorectal cancer (CRC) is known as the most common gastrointes- tinal malignancy and one of the leading causes of cancer death world- wide [1, 2]. The survival of CRC patients has been improved by radical re- section combined with adjuvant chemoradiotherapy [3], but recurrence or distant metastasis of CRC occurs in most patients and is the primary cause of treatment failure [4]. Therefore, a suitable indicator of the prog- nosis of CRC patients is pivotal to treatment in the clinic. Bo Sun, Jun Wang, Yan-Feng Zhu, Zhen-Yang Li, Jian-Bin Xiang, Zong-You Chen, Zhi-Gang He, Xiao-Dong Gu Translocase of the inner mitochondrial mem- Immunohistochemistry analysis of TIMM50 brane 50 (TIMM50) is an essential component Immunohistochemistry (IHC) was performed of the TIM23 complex, which mediates the trans- strictly in accordance with the manufacturer’s location of transit peptide-containing proteins instructions. In brief, tissue sections were cut to across the mitochondrial inner membrane [5, 6]. 3-μm thickness and placed on slides. The slides In humans, two isoforms of TIMM50 have been were incubated for 1 h at 72°C, immersed in xy- reported: a short TIMM50S (353 amino acids) lene twice and rehydrated with graded ethanol. isoform and a long TIMM50L (456 amino acids) The slides were washed with PBS 5 times. The sec- isoform. TIMM50S exists in mitochondria as part tions were immersed in sodium citrate buffer (pH of the TIM23 complex, whereas TIMM50L targets 6.0) for 30 min at 95°C to enhance antigen re- the nucleus due to the presence of an internal nu- trieval. The sections were placed in 3% hydrogen clear localization signal [7–9]. TIMM50 has been peroxide for 10 min at room temperature to block studied in tumour progression. Gao et al. [10] re- endogenous peroxidase activity. Then, the slices vealed that loss of TIMM50 suppressed cancer cell were incubated for 24 h with a specific primary proliferation and induced cancer cell apoptosis in antibody for TIMM50 (1 : 100 dilution; Santa Cruz breast cancer. Zhang et al. [11] also found that Biotechnology Inc., Santa Cruz, USA). After wash- overexpression of TIMM50 promoted tumour pro- ing with PBST buffer, the slices were incubated gression by phosphorylating ERK/P90RSK in non- with a goat anti-mouse biotinylated antibody for small cell lung cancer cells, and they predicted 1 h. Colour development was carried out using TIMM50 to be a prognostic marker in non-small 3,3ʹ-diaminobenzidine (DAB), and counterstaining cell lung cancer patients. Nevertheless, whether was performed using haematoxylin. After stain- TIMM50 is a biomarker for predicting the progno- ing, the sections were dehydrated with increasing sis of CRC patients remains unknown. concentrations of ethanol and xylene. To investigate the prognostic role of TIMM50 The staining results were evaluated by two ex- expression and assess the potential connection perienced pathologists in a double-blind manner. between TIMM50 expression and clinicopatholo- A semi-quantitative method was applied to judge gical characteristics in CRC, we performed immu- the percentage of positive cells under the mi- nohistochemistry on 192 CRC patient samples; we croscope and the staining intensity. The number found that TIMM50 may be a sensitive biomarker of positive cells was observed in 5 high power for the prognosis of CRC patients. fields (×200) for each slice, and the percentage of positive cells was counted. Positive colour in- Material and methods tensity was scored as 0 (normal staining), 1 (weak Patients and tissue samples staining), 2 (intermediate staining), and 3 (strong staining). The percentage of positive cells was Colorectal cancer patients (123 men and 69 scored as 1 (< 25%), 2 (26–50%), 3 (51–75%) and women) who had undergone radical resection 4 (> 75%) [3]. The two scores were multiplied to from May 2013 to April 2018 participated in this obtain the final score. TIMM50 expression was de- study. None of the patients received chemoradio- fined as low for a score < 6 and high for a score > 6. therapy prior to surgery. The patients with colon cancers underwent the same surgical procedure Quantitative real-time PCR for complete mesocolic excision. The patients with rectal cancers underwent the same surgical proce- Total RNA was extracted from tumour tissues and dure for total mesorectal excision. This study was adjacent tissues with Trizol homogenization buffer approved by the Ethics Committee of Huashan (Takara Bio. Inc., Takara, Japan); then, according Hospital. Written consent was provided by all par- to the PrimeScript RT reagent kit protocol (Takara ticipants in this study. Tumour and normal tissues Bio. Inc.), RNA was reverse transcribed into cDNA. were procured from patients involved in this study. The cDNA was amplified by quantitative real-time The tissues were snap-frozen after resection and PCR using specific primers for TIMM50 (forward: stored in liquid nitrogen. The patients were fol- 5ʹ-TTCCTGATGAGTTCGACAATG-3ʹ; reverse: 5ʹ-AGCTC- lowed up every three months after surgery for CAAAACGAGCGTGTA-3ʹ) and GAPDH (forward: five years. During follow-up, regular evaluations 5’-CTGACTTCAACAGCGACACC-3ʹ; reverse: 5ʹ-TAG included medical history, physical examination, CCA A ATTCGTTGTCATACC-3ʹ). The reaction volume complete blood count, thoracic computed tomog- was 20 μl (including 10 μl of SYBR green mix, 0.5 μl raphy (CT), and abdominal ultrasound or CT. Dis- of each primer, 1 μl of cDNA, and 8 μl of ddH2O), and ease progression was determined by the treating samples were detected with an ABI 7500 Real-Time physician based on the available information, in- PCR system. GAPDH was used as an internal refer- cluding clinical assessments, radiologic examina- ence gene, and the relative TIMM50 expression was tion, and pathology reports. determined using the Livak method (2–ΔΔCt). 2 Arch Med Sci Prognostic value of TIMM50 expression in colorectal cancer Western blotting Results The tumour and adjacent tissue samples were Immunohistochemical staining lysed completely in extraction buffer. The super- characteristics natants were centrifuged and collected. The pro- The samples were from 192 patients from our tein concentrations were determined according hospital. The expression of TIMM50 was detected to the BCA protocol. Equal amounts of protein by immunohistochemical staining. TIMM50 ex- samples (30 μg) were separated by 10% SDS- pression was assessed and scored. The TIMM50 PAGE. Then, the proteins were transferred onto immunohistochemical staining characteristics for polyvinylidene fluoride (PVDF) membranes. colorectal cancers were as follows: staining inten- Next, the PVDF membranes were incubated with sity was 0 (Figure 1A), 1 (Figure 1B), 2 (Figure 1C) primary antibodies at 4°C overnight, including and 3 (Figure 1D). The percentage of positive TIMM50 and GAPDH (1 : 1000, Santa Cruz Biotech- cells was designated as 1 (1–25%) (Figure 1E), nology Inc., Santa Cruz,
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