王 哲 張 敬 陳懷安 張 潮 劉 碩 苗文隆

(河北北方學院附屬第一醫(yī)院泌尿外科,張家口 075000)

膀胱癌是臨床常見惡性腫瘤之一，因其具有高轉(zhuǎn)移率及復(fù)發(fā)率的特點而備受關(guān)注[1,2]。但膀胱癌發(fā)病機制尚不明確，導致目前仍無治療藥物能明顯影響其預(yù)后[3]。因此，探究膀胱癌發(fā)病機制是尋找治療膀胱癌新藥及新方法的關(guān)鍵。已有研究表明長鏈非編碼RNA UCA1在膀胱癌中呈高表達狀態(tài)，并且能夠促進膀胱癌細胞的侵襲遷移，其機制與調(diào)控其下游microRNAs(miRNAs)的表達有關(guān)[4-6]。miR-582-5p 是一類抑癌基因，上調(diào)miR-582-5p表達可明顯抑制肝癌、結(jié)腸癌及前列腺癌等癌細胞增殖[7-9]。 也有研究表明，miR-582-5p低表達也與膀胱癌的發(fā)展密切相關(guān)[10]。并且生物信息預(yù)測表明UCA1可能與miR-582-5p之間存在靶向關(guān)系。但UCA1能否通過靶向調(diào)控miR-582-5p的表達影響膀胱癌的增殖和遷移能力還未見報道。因此本文將以膀胱癌UM-UC-3為對象，探討UCA1對膀胱癌細胞生存及運動能力的作用及作用機制。

1 材料與方法

1.1材料

1.1.1試劑及細胞 MEM細胞培養(yǎng)液、胎牛血清、胰酶和轉(zhuǎn)染試劑盒均購自美國Invitrogen公司;Matrigel購自美國BD公司；TRIzol試劑盒、反轉(zhuǎn)錄試劑盒和熒光定量試劑盒購自美國ThermoFisher公司；Ki67、cleaved caspase-3和VEGF一抗購自美國Millipore公司；HRP標記的山羊抗小鼠二抗購自美國Santa Cruz公司；人膀胱癌細胞UM-UC-3購自美國ATCC，貨號為CRL-1749TM；UCA1 shRNA(sh-UCA1)由上海生工生物公司設(shè)計合成，miR-582-5p inhibitor購自美國Invitrogen公司。

1.1.2儀器 ELX808酶標儀購自美國Bio-Tek公司。BD LSRFortessaTM流式細胞儀購自美國Biosciences公司。PCR儀、電泳儀及半干轉(zhuǎn)膜儀均購自美國伯樂公司。Gel View 6000化學發(fā)光凝膠成像系統(tǒng)購自廣州云星儀器有限公司。

1.2方法

1.2.1細胞培養(yǎng) 人膀胱癌細胞用含10% 胎牛血清的MEM培養(yǎng)液于37℃ 5% CO2的培養(yǎng)箱中培養(yǎng)，隔天換液。細胞融合率達到85%時進行傳代培養(yǎng)。

1.2.2細胞轉(zhuǎn)染 將細胞接種于6孔板中。培養(yǎng)24 h后根據(jù)轉(zhuǎn)染試劑盒說明書用Lipofectamine 2000分別或同時轉(zhuǎn)染sh-UCA1和miR-582-5p inhibitor，48 h后進行相應(yīng)檢測。

1.2.3CCK8檢測細胞活性 將UM-UC-3細胞傳代培養(yǎng)于96孔板中，培養(yǎng)24 h后將細胞隨機分為sh-Ctrl組、sh-UCA1組、sh-UCA1+miR-582 mock組和sh-UCA1+miR-582 inhib組，對細胞進行相應(yīng)轉(zhuǎn)染后，根據(jù)試劑盒說明書每孔加入10 μl CCK8試劑并于培養(yǎng)箱中繼續(xù)孵育2 h，用酶標儀檢測各組吸光度，計算細胞生長速度(增殖倍數(shù)=細胞吸光度/0 h 細胞吸光度)。

1.2.4流式細胞術(shù)檢測細胞凋亡 細胞轉(zhuǎn)染后48 h，用胰酶收集細胞，將細胞以1 000 r/min轉(zhuǎn)速離心4 min，用緩沖液清洗3次后重懸沉淀，使細胞密度為3×106個/ml。加入FITC-Annexin V和PI溶液，避光孵育15 min后用流式細胞儀檢測細胞凋亡情況。

1.2.5劃痕實驗 實驗前用Maker筆于培養(yǎng)板背面劃平行的5條直線，滅菌后備用。將細胞傳代培養(yǎng)于12孔板中，用sh-UCA1和miR-582-5p inhibitor轉(zhuǎn)染后，用10 μl槍頭垂直于培養(yǎng)板背面直線劃痕，用預(yù)冷的PBS洗滌3次后，加入無血清培養(yǎng)液進行培養(yǎng)，于0 h、24 h進行拍照記錄，并計算劃痕愈合率[劃痕閉合率 =(0 h劃痕寬度-24 h劃痕寬度)/0 h劃痕寬度]×100%。

1.2.6侵襲實驗 將膀胱癌細胞以無血清培養(yǎng)液培養(yǎng)24 h后，用0.25%胰蛋白酶消化細胞并傳代接種于用Matrigel預(yù)處理的Transwell小室中，細胞密度為3×105個/ml。小室上層加入無血清培養(yǎng)液培養(yǎng)細胞，下層則加入含血清的正常培養(yǎng)液。48 h后用無菌棉簽擦去小室上層細胞，下層細胞HE染色后計數(shù)統(tǒng)計。

1.2.7熒光素酶報告實驗 首先通過生物信息預(yù)測miR-582-5p在UCA1上的結(jié)合位點并用PCR對UCA1上miR-582-5p的結(jié)合位點進行擴增。隨后將擴增片段插入到pMIR-REPORT載體，構(gòu)建UCA1野生型質(zhì)粒。用基因突變技術(shù)將部分核苷酸突變，構(gòu)建UCA1變型質(zhì)粒。用UCA1野生型質(zhì)粒、UCA1變型質(zhì)粒和miR-582 mimic轉(zhuǎn)染細胞后，用熒光素酶檢測試劑盒進行檢測，最后檢測各組熒光素酶活性。

1.2.8qRT-PCR 用TRIzol試劑盒提取各組細胞總RNA。根據(jù)逆轉(zhuǎn)錄試劑盒說明書合成cDNA，用PCR儀對cDNA進行擴增后用熒光定量試劑盒對進行定量分析。以β-actin為內(nèi)參。反應(yīng)條件為：95℃ 反應(yīng)10 s、5 s，總共40個循環(huán)，最后60℃反應(yīng)30 s，70℃反應(yīng)30 s。實驗所用的引物序列如下：UCA1 上游引物：5′-CTCTCCATTGGGTTCACCATTC-3′，下游引物：5′-GCGGCAGGTCTTAAGAGATGAG-3′；miR-582-5p上游引物：5′-GCACACATTGAAGAGG-ACAGAC-3′，下游引物：5′-TATTGAAGGGGGTT-CTGGTG-3′，β-actin 上游引物：5′-GGCACCACCATGTACCCTG-3′，下游引物：5′-CACGGAGTACTTGC-GCT CAG-3′。

1.2.9Western blot 用RIPA裂解液提取各組細胞總蛋白。用BCA試劑盒檢測總蛋白濃度，10% SDS-PAGE分離蛋白后用半干轉(zhuǎn)膜儀轉(zhuǎn)移蛋白質(zhì)至PVDF膜。用5%脫脂牛奶室溫封閉蛋白2 h，隨后加入一抗(Ki67,1∶1 000;cleaved caspase-3,1∶1 000;VEGF,1∶1 000)于4℃封閉過夜，第2天加入對應(yīng)二抗室溫封閉1 h，最后滴加ECL曝光顯影。

2 結(jié)果

2.1沉默UCA1對膀胱癌細胞miR-582-5p表達的影響 實驗結(jié)果表明，sh-UCA1轉(zhuǎn)染細胞后，sh-UCA1組UM-UC-3細胞UCA1表達水平較sh-Ctrl組比較明顯降低，表明轉(zhuǎn)染成功(P<0.05，圖1)；與sh-Ctrl組比較，sh-UCA1組和sh-UCA1+miR-mock組miR-582-5p表達水平明顯升高(P<0.05，圖1)；與sh-UCA1+miR-mock組比較，sh-UCA1+miR-inhib組細胞miR-582-5p表達水平明顯降低(P<0.05，圖1)。

2.2UCA1與miR-582-5p的靶向關(guān)系 生物信息預(yù)測結(jié)果表明，UCA1上存在miR-582-5p的結(jié)合位點。因此，本研究采用熒光素酶報告實驗進一步確定UCA1和miR-582-5p的靶向關(guān)系。實驗結(jié)果表明，miR-582 mimic可顯著降低UCA1野生型質(zhì)粒熒光素酶的活性(P<0.05，圖2)；結(jié)合位點突變后，miR-582 mimic對UCA1質(zhì)粒熒光素酶活性的調(diào)控關(guān)系消失，表明UCA1上存在miR-582-5p的結(jié)合位點。

2.3沉默UCA1對膀胱癌細胞生存能力的影響 實驗結(jié)果表明，與sh-Ctrl組比較，細胞轉(zhuǎn)染后5 d，sh-UCA1組和sh-UCA1+miR-mock組膀胱癌細胞生長速度明顯降低(P<0.05，圖3)；sh-UCA1+miR-inhib組細胞生長速度與sh-UCA1+miR-mock組比較顯著升高，差異有統(tǒng)計學意義(P<0.05，圖3)。

圖1 沉默UCA1對膀胱癌細胞miR-582-5p表達的影響Fig.1 Effect of silencing UCA1 on expression of miR-582-5p in bladder cancer cellsNote:Cells were transferred with sh-UCA1 and miR-582-5p inhibitor.mRNA levels of UCA1 and miR-582-5p were measured by RT-PCR.n=6,β-actin was used as loading control.*.P<0.05 versus sh-Ctrl group;#.P<0.05 versus sh-UCA1+miR-mock group.

同時，sh-UCA1組和sh-UCA1+miR-mock組細胞增殖標記蛋白Ki67表達水平與sh-Ctrl組比較明顯降低(P<0.05，圖4)；sh-UCA1+miR-inhib組細胞Ki67的表達明顯升高，與sh-UCA1+miR-mock組比較差異有統(tǒng)計學意義(P<0.05，圖4)；此外，sh-UCA1還能顯著誘導膀胱癌細胞凋亡，促進凋亡標記蛋白cleaved caspase-3表達(P<0.05，圖4、5)； miR-582-5p inhibitor能明顯減弱sh-UCA1對膀胱癌細胞凋亡和cleaved caspase-3表達的促進作用(P<0.05，圖4、5)。

2.4沉默UCA1對膀胱癌細胞運動能力的影響為了進一步探究UCA1通過靶向miR-582-5p對UM-UC-3細胞運動能力的影響，我們用Transwell實驗和劃痕實驗檢測sh-UCA1和miR-582-5p inhibitor轉(zhuǎn)染后細胞的侵襲及遷移能力。Transwell實驗結(jié)果表明，與sh-Ctrl組比較，sh-UCA1組和sh-UCA1+miR-mock組細胞侵襲數(shù)明顯減少(P<0.05，圖6)；與sh-UCA1+miR-mock組比較，sh-UCA1+miR-inhib組細胞侵襲數(shù)目明顯增多(P<0.05，圖6)；同時，細胞轉(zhuǎn)染48 h后，sh-UCA1組和sh-UCA1+miR-mock組劃痕閉合程度明顯低于sh-Ctrl組(P<0.05， 圖7)，sh-UCA1+miR-inhib組劃痕閉合程度較sh-UCA1+miR-mock組明顯升高，差異有統(tǒng)計學意義(P<0.05，圖7)。此外，sh-UCA1還能顯著抑制VEGF的表達(P<0.05，圖4)；下調(diào)miR-582-5p表達則能明顯減弱sh-UCA1對VEGF表達的抑制作用(P<0.05，圖4)。

圖2 UCA1與miR-582-5p的靶向關(guān)系Fig.2 Relationship of UCA1 and miR-582-5pNote:Luciferase reporter assay was performed for targeted-relationship of UCA1 and miR-582-5p.*.P<0.05 versus miR-582 mock group;#.P<0.05 versus UCA1 wt group.

圖3 沉默UCA1對膀胱癌細胞生長的影響Fig.3 Effect of silencing UCA1 on growth of bladder cancer cellsNote:Cell viability was measured by CCK8 assay after cells were transferred with sh-UCA1 and miR-582-5p inhibitor.*.P<0.05 versus sh-Ctrl group;#.P<0.05 versus sh-UCA1+miR-mock group.

圖4 沉默UCA1對膀胱癌細胞增殖、凋亡及侵襲遷移相關(guān)蛋白表達的影響Fig.4 Effects of silencing UCA1 on expression levels of proliferation-,apoptosis-and migration-related proteinsNote:Western blot was performed for protein levels of Ki67，cleaved caspase-3 and VEGF.GAPDH was used as loading control.*.P<0.05 versus sh-Ctrl group;#.P<0.05 versus sh-UCA1+miR-mock group.

圖5 沉默UCA1對膀胱癌細胞凋亡的影響Fig.5 Effect of UCA1 silencing on apoptosis of bladder caner cellsNote:Apoptosis was determined by flow cytometry.*.P<0.05 versus sh-Ctrl group;#.P<0.05 versus sh-UCA1+miR-mock group.

圖1 沉默UCA1對膀胱癌細胞侵襲能力的影響。

圖7 沉默UCA1對膀胱癌細胞遷移能力的影響Fig.7 Effect of silencing UCA1 on migration ability of bladder cancer cellsNote:Wound healing assay was performed for migration ability.*.P<0.05 versus sh-Ctrl group;#.P<0.05 versus sh-UCA1+miR-mock group.

3 討論

大量研究表明，長鏈非編碼RNA在癌癥的發(fā)展和轉(zhuǎn)移過程中發(fā)揮重要作用[11,12]。長鏈非編碼RNA UCA1是一類癌癥誘導基因，其在多類癌癥中均呈高表達狀態(tài)，如乳腺癌、結(jié)腸癌、胃癌和肝癌等[6,12-15]。其在多類膀胱癌細胞系中表達顯著升高，促進膀胱癌細胞增殖和遷移[16,17]。本研究發(fā)現(xiàn)UCA1在膀胱癌細胞UM-UC-3中呈高表達狀態(tài)，且與癌癥的發(fā)生發(fā)展有關(guān)。

研究表明，非編碼RNA發(fā)揮生物學功能與抑制或上調(diào)下游靶標miRNA的表達密切相關(guān)[18-20]。UCA1可通過調(diào)控多種miRNA的表達影響癌癥的發(fā)展。但UCA1是否能通過靶向調(diào)控抑癌基因miR-582-5p的表達影響膀胱癌的惡性進展未見報道。本研究發(fā)現(xiàn)miR-582-5p在膀胱癌細胞中表達明顯降低，這也與之前的報道一致[10]。沉默UCA1能顯著上調(diào)膀胱癌細胞miR-582-5p的表達。用miR-582-5p inhibitor抑制miR-582-5p表達后，sh-UCA1對miR-582-5p表達的促進作用明顯減弱，提示UCA1和miR-582-5p之間可能存在靶向調(diào)控關(guān)系。生物信息預(yù)測結(jié)果表明，UCA1上存在miR-582-5p的結(jié)合位點，熒光素酶報告實驗發(fā)現(xiàn)，miR-582-5p過表達可明顯減弱UCA1野生質(zhì)粒的熒光素酶活性，進一步表明miR-582-5p可與UCA1結(jié)合，UCA1與miR-582-5p之間存在靶向調(diào)控關(guān)系。

癌細胞無限增殖和細胞凋亡抑制是癌癥發(fā)生發(fā)展的重要機制。已有研究表明UCA1能明顯促進膀胱癌細胞增殖、誘導癌細胞凋亡[21]。本文研究發(fā)現(xiàn)，下調(diào)UCA1表達能顯著抑制膀胱癌細胞增殖，還可降低細胞增殖標記蛋白Ki67的蛋白表達水平，而通過抑制miR-582-5p表達可部分恢復(fù)UCA1沉默引起的膀胱癌細胞的增殖受抑，說明UCA1促進膀胱癌細胞增殖與其抑制miR-582-5p表達有關(guān)。此外，沉默UCA1還能誘導膀胱細胞凋亡，促進cleaved caspase-3表達，miR-582-5p inhibitor可明顯減弱sh-UCA1對膀胱癌細胞凋亡的作用，提示UCA1調(diào)控膀胱癌細胞凋亡與其靶向調(diào)控miR-582-5p表達有關(guān)。

癌細胞轉(zhuǎn)移是導致癌癥惡化和癌癥患者死亡的主要原因[22,23]。抑制癌細胞轉(zhuǎn)移能明顯延長癌癥患者的生存時間，降低癌癥術(shù)后復(fù)發(fā)率[24,25]。UCA1過表達能促進癌細胞轉(zhuǎn)移[26,27]，可被作為惡性腫瘤轉(zhuǎn)移和不良預(yù)后的指標[28]。本文研究發(fā)現(xiàn)，沉默UCA1表達能明顯降低膀胱癌細胞的侵襲及遷移能力，并能抑制VEGF的表達。VEGF是血管新生重要調(diào)控分子，其可通過促進細胞外基質(zhì)降解促進細胞的遷移，促進VEGF表達可促進膀胱癌細胞遷移[29]。同時，抑制miR-582-5p表達能明顯減弱sh-UCA1對膀胱癌細胞侵襲、遷移的抑制作用，提示UCA1促進膀胱癌細胞轉(zhuǎn)移與其下調(diào) miR-582-5p的表達有關(guān)。

綜上所述，沉默UCA1能明顯抑制膀胱癌細胞增殖、侵襲和遷移，同時還能誘導膀胱癌細胞凋亡，減緩膀胱癌的惡性進展。抑制miR-582-5p表達能明顯減弱sh-UCA1的抑癌作用，表明UCA1可通過靶向miR-582-5p增強膀胱癌細胞的生存及運動能力。本研究進一步闡明了UCA1促進膀胱癌發(fā)展的機制，可能為膀胱癌的治療提供了又一新的靶標。

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