Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.
Department of Histopathology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.
Indian J Med Res. 2017 Oct;146(4):520-527. doi: 10.4103/ijmr.IJMR_761_15.
BACKGROUND & OBJECTIVES: Coxsackievirus B (CVB), a member of human Enterovirus group, is the most common cause of viral myocarditis. Coxsackievirus adenovirus receptor (CAR) is identified as a key determinant for the entry of CVB in the target cells. Thus, blockade of receptor by RNA interference (RNAi) may inhibit the entry and pathogenesis of CVB in cardiac cells. The present study was aimed to determine the effect of CAR small dsRNA (siRNA) on coxsackieviral load and CAR expression in coxsackievirus-infected cardiomyocytes.
Transfection efficiency in rat cardiomyocytes (H9c2) was determined by the fluorescent microscopy and flow cytometry. CAR siRNA dose was optimized based on cell viability and relative CAR messenger RNA (mRNA) expression. Cardiomyocytes were transfected with CAR siRNA followed by infection with 100 multiplicity of infection of CVB, which were harvested after 24, 48 and 72 h post-infection (p.i.). RNA was extracted for relative CAR mRNA expression. Cells were freeze-thawed thrice for estimating coxsackieviral load.
The efficiency of transfection was optimized to be >80 per cent and CAR siRNA dose of 60 pmol was standardized. The knockdown of CAR by siRNA decreased its expression twice the expression in normal cardiomyocytes after 24 h p.i. of CVB. The treatment with CAR siRNA resulted in significant two log reduction of CVB load in cardiomyocytes infected with CVB at 24 h p.i. and retained till 72 h p.i.
INTERPRETATION & CONCLUSIONS: The inhibition of CAR by siRNA was found to be effective against CVB in cardiomyocytes. However, this treatment strategy has to be evaluated in vivo to develop a new treatment strategy for patients suffering with viral myocarditis.
柯萨奇病毒 B（CVB）是人肠道病毒属的成员，是病毒性心肌炎的最常见病因。柯萨奇病毒腺病毒受体（CAR）被鉴定为 CVB 进入靶细胞的关键决定因素。因此，通过 RNA 干扰（RNAi）阻断受体可能会抑制 CVB 在心肌细胞中的进入和发病机制。本研究旨在确定 CAR 小 dsRNA（siRNA）对感染柯萨奇病毒的心肌细胞中柯萨奇病毒载量和 CAR 表达的影响。
通过荧光显微镜和流式细胞术确定大鼠心肌细胞（H9c2）的转染效率。根据细胞活力和相对 CAR 信使 RNA（mRNA）表达优化 CAR siRNA 剂量。用 CAR siRNA 转染心肌细胞，然后用 100 感染复数的 CVB 感染，感染后 24、48 和 72 小时收获细胞。提取 RNA 以检测相对 CAR mRNA 表达。细胞经冻融 3 次以估计柯萨奇病毒载量。
优化转染效率>80%，并确定 CAR siRNA 剂量为 60 pmol。siRNA 对 CAR 的敲低使感染 CVB 24 小时后 CAR 的表达降低至正常心肌细胞的两倍。在感染 CVB 24 小时时，CAR siRNA 的治疗导致心肌细胞中 CVB 载量显著降低两个对数级，并在 72 小时时仍保留。
发现 siRNA 抑制 CAR 对心肌细胞中的 CVB 有效。然而，这种治疗策略必须在体内进行评估，以开发治疗病毒性心肌炎患者的新治疗策略。
