蔣小麗，周慧芳，毛能建

新疆維吾爾自治區(qū)喀什地區(qū)第一人民醫(yī)院檢驗(yàn)科(喀什844000)

miR-425調(diào)控PDL-1通路調(diào)節(jié)腎上皮細(xì)胞增殖、凋亡作用的實(shí)驗(yàn)研究*

蔣小麗，周慧芳，毛能建

新疆維吾爾自治區(qū)喀什地區(qū)第一人民醫(yī)院檢驗(yàn)科(喀什844000)

目的：探討miR-425通過調(diào)控PDL-1通路調(diào)節(jié)腎上皮細(xì)胞的增殖及凋亡作用。方法：腎上皮細(xì)胞HRCEpiC分別轉(zhuǎn)染miR-425模擬物及對(duì)照模擬物，采用qRT-PCR方法檢測(cè)miR-425表達(dá)水平，采用MTT法與Tunel試驗(yàn)測(cè)定細(xì)胞增殖及凋亡作用，采用Western blot實(shí)驗(yàn)測(cè)定PDL-1表達(dá)水平。結(jié)果： qRT-PCR結(jié)果顯示轉(zhuǎn)染后實(shí)驗(yàn)組miR-425的表達(dá)水平較空白組、陰性對(duì)照組升高約37.82倍。轉(zhuǎn)染48 h后實(shí)驗(yàn)組、陰性對(duì)照組與空白組的活細(xì)胞比例分別為(75.22±3.19)%、(98.29±2.11)%和(100±0.00)%，細(xì)胞凋亡率分別為(17.20±2.49)%、(4.29±1.44)%和(0.52±0.11)%，組間對(duì)比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；轉(zhuǎn)染48 h后Western blot檢測(cè)顯示實(shí)驗(yàn)組的PDL-1表達(dá)水平都明顯低于陰性對(duì)照組與空白組(P<0.05)。結(jié)論：miR-425可通過激活PDL-1通路，降低腎上皮細(xì)胞增殖能力，提高其凋亡能力，從而介導(dǎo)腎上皮細(xì)胞的生物學(xué)行為。

材料與方法

1 材 料 腎上皮細(xì)胞HRCEpiC源于中國醫(yī)學(xué)科學(xué)院細(xì)胞中心，選擇RPMI1640+10%血清進(jìn)行培養(yǎng)(5%CO2、37℃孵箱)；miR-425模擬物及對(duì)照模擬物購自Ambion 公司；免疫印跡化學(xué)發(fā)光(ECL)系統(tǒng)購自美國Syngene公司；兔抗人β-actin單克隆抗體購自Abcam公司，兔抗人PDL-1單克隆抗體及相應(yīng)二抗及熒光二抗均購自Santa Cruz公司；脂質(zhì)體Lipo-fectamine2000購自Invitrogen公司；MTT檢測(cè)試劑盒購自美國Sigma 公司。

2 實(shí)驗(yàn)方法 ①細(xì)胞增殖實(shí)驗(yàn)：選擇HRCEpiC細(xì)胞中通過脂質(zhì)體轉(zhuǎn)染試劑瞬時(shí)轉(zhuǎn)染miR-425模擬物及對(duì)照模擬物，并通過Real time PCR分別驗(yàn)證轉(zhuǎn)染效率。將轉(zhuǎn)染后的細(xì)胞株分別以5000個(gè)/孔的密度接種于96空培養(yǎng)板中，培養(yǎng)48 h后，應(yīng)用MTT試劑盒測(cè)定細(xì)胞增殖性，各實(shí)驗(yàn)均重復(fù)接種于3個(gè)孔中。②細(xì)胞凋亡實(shí)驗(yàn)：采用原位細(xì)胞凋亡檢測(cè)技術(shù)(Tunel試驗(yàn))，HRCEpiC細(xì)胞在96孔培養(yǎng)48 h后取1 ml細(xì)胞，1000 r/min，4 ℃離心10 min，取沉淀，PBS重懸細(xì)胞，離心后取呈現(xiàn)，加入300 μl凋亡檢測(cè)緩沖液，以每個(gè)象限的細(xì)胞數(shù)目是檢測(cè)細(xì)胞總數(shù)所在點(diǎn)的百分比計(jì)算凋亡率，各實(shí)驗(yàn)均重復(fù)接種于3個(gè)孔中。③Western blot實(shí)驗(yàn)：在miR-425瞬時(shí)轉(zhuǎn)染分別在48 h 后提取細(xì)胞總蛋白，并用BCA法測(cè)定蛋白濃度。各組取等量濃度的蛋白樣本，進(jìn)行SDS-PAGE 凝膠電泳，然后將蛋白轉(zhuǎn)移至PVDF 膜上，封閉后加入一抗PDL-1，4 ℃過夜。TBST洗膜30 min，加入二抗室溫孵育1 h，TBST 洗膜30 min，加入ECL發(fā)光劑，進(jìn)行電腦掃描發(fā)光。

結(jié) 果

1 轉(zhuǎn)染效果 腎上皮細(xì)胞HRCEpiC分別轉(zhuǎn)染miR-425模擬物為實(shí)驗(yàn)組，轉(zhuǎn)染對(duì)照模擬物為陰性對(duì)照組，轉(zhuǎn)染組為空白組。qRT-PCR結(jié)果顯示轉(zhuǎn)染后實(shí)驗(yàn)組miR-425的表達(dá)水平較空白組、陰性對(duì)照組升高約37.82倍，見表1。

表1 miR-425轉(zhuǎn)染后的ct值對(duì)比

2 細(xì)胞增殖情況對(duì)比 轉(zhuǎn)染48 h后實(shí)驗(yàn)組、陰性對(duì)照組與空白組的活細(xì)胞比例分別為(75.22±3.19)%、(98.29±2.11)%和(100±0.00)%，組間對(duì)比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

3 細(xì)胞凋亡情況對(duì)比 轉(zhuǎn)染48 h后實(shí)驗(yàn)組、陰性對(duì)照組與空白組的細(xì)胞凋亡率分別為(17.20±2.49)%、(4.29±1.44)%和(0.52±0.11)%，組間對(duì)比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

4 PDL-1表達(dá)對(duì)比 轉(zhuǎn)染48 h后Western blot檢測(cè)顯示，實(shí)驗(yàn)組的PDL-1表達(dá)水平(0.51±0.01)明顯低于陰性對(duì)照組(0.65±0.04)與空白組(0.69±0.06)(P<0.05)。

討 論

腎上皮細(xì)胞的作用是多方面的，首先上皮細(xì)胞通過不斷的增殖，對(duì)損傷的腎組織進(jìn)行修復(fù)，維持上皮細(xì)胞屏障結(jié)構(gòu)的完整性[4]。另外上皮細(xì)胞的效應(yīng)進(jìn)一步擴(kuò)展為潛在的直接免疫調(diào)節(jié)作用，且這種作用與炎癥刺激時(shí)間之間具有一定的相關(guān)性[5]。比如HRCEpiC能產(chǎn)生IL-8或直接趨化白細(xì)胞參與急性炎癥反應(yīng)，可參與腎免疫損傷的發(fā)生[6]。

MicroRNA是一類21-23 nt的非編碼小分子RNA，它們通過與靶mRNA的編碼區(qū)、3'-UTR完全或部分配對(duì)，降解靶mRNA或抑制其蛋白質(zhì)翻譯，在轉(zhuǎn)錄后水平負(fù)調(diào)控靶基因的表達(dá)[7]。miR-425家族在上皮細(xì)胞的形成過程中發(fā)揮重要作用，抑制上皮細(xì)胞形成過程，促進(jìn)E-鈣黏蛋白表達(dá)[8]。本研究顯示轉(zhuǎn)染后實(shí)驗(yàn)組、陰性對(duì)照組與空白組的活細(xì)胞比例分別為(75.22±3.19)%、(98.29±2.11)%和(100±0.00)%，組間對(duì)比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；轉(zhuǎn)染48 h后實(shí)驗(yàn)組、陰性對(duì)照組與空白組的細(xì)胞凋亡率分別為(17.20±2.49)%、(4.29±1.44)%和(0.52±0.11)%，組間對(duì)比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，表明miR-425表達(dá)與腎組織細(xì)胞增殖能力呈負(fù)相關(guān)，與凋亡能力成正相關(guān)，miR-425可能參與腎上皮細(xì)胞的調(diào)控。

PD1最有兩個(gè)配體，分別是PDL-1(B7-H1)和PDL-2(B7-DC)，PDL-1蛋白廣泛表達(dá)于抗原提呈細(xì)(APCs)、活化T、B細(xì)胞等。PDL-1與受體結(jié)合后可誘導(dǎo)內(nèi)皮細(xì)胞生長(zhǎng)、增殖，促進(jìn)內(nèi)皮細(xì)胞遷移[9]。本研究轉(zhuǎn)染48 h后Western blot檢測(cè)顯示，實(shí)驗(yàn)組的PDL-1表達(dá)水平都明顯低于陰性對(duì)照組與空白組(P<0.05)，表明miR-425可能參與PDL-1信號(hào)轉(zhuǎn)導(dǎo)通路，從而參與調(diào)控腎組織的增殖與凋亡。

總之，miR-425可通過激活PDL-1通路，降低腎上皮細(xì)胞增殖能力，提高其凋亡能力，從而介導(dǎo)腎上皮細(xì)胞的生物學(xué)行為。

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[5] Massari F，Santoni M，Ciccarese C，etal. PD-1 blockade therapy in renal cell carcinoma: current studies and future promises[J].Cancer Treat Rev，2015，41(2):114-121.

[6] Joseph RW，Parasramka M，Eckel-Passow JE，etal.Inverse association between programmed death ligand 1 and genes in the VEGF pathway in primary clear cell renal cell carcinoma[J].Cancer Immunol Res，2013，1(6):378-385.

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miR-425regulatestheproliferationandapoptosisofrenalepithelialcellbyregulatingPDL-1pathway

Jiang Xiaoli，Zhou Huifang，Mao Nengjian.

Department of Laboratory，The First People’s Hospital of Kashgar Region，Kesha Xinjiang(Kashgar 844000)

Objective: To investigate the effects of miR-425 regulates the proliferation and apoptosis of renal epithelial cell by regulating PDL-1 pathway.Methods: Nasal polyp epithelial cells HRCEpiC were transfected miR-425 mimic and control，qRT-PCR method was used to detect the expression level of miR-425，MTT method and Tunel test were used to detect the cell proliferation and apoptosis，the level of PDL-1 expression were determined by Western blot experiment. Results：The results of qRT-PCR showed that the expression level of miR-425 in the experimental group was about 37.82 times higher than that in the blank group and the negative control group. After transfection of 48h，the proportion of live cells in the experimental group，negative control group and blank group were (75.22±3.19)%，(98.29±2.11)% and (100±0.00)%，the apoptosis rate were(17.20±2.49)%，(4.29±1.44)% and (0.52±0.11)%，there were statistically significant difference compared among the groups (P<0.05); Western blot assay showed that transfection after 48 h，the expression level of PDL-1 in the experimental group were significantly lower than the negative control group and blank control group (P<0.05).Conclusion: miR-425 can inhibit the proliferation of nasal polyps epithelial cells by increasing PDL-1 pathway，and improve the ability of apoptosis.

Renal epithelial cells Cell proliferation Apoptosis @miR-425 @PDL-1

*新疆維吾爾自治區(qū)自然科學(xué)基金資助項(xiàng)目(2016D01C022)

腎上皮細(xì)胞 細(xì)胞增殖 細(xì)胞凋亡 @miR-425 @PDL-1

R623.1

A

10.3969/j.issn.1000-7377.2017.10.006

(收稿：2017-04-01)