[摘要] 目的
探討circPCMTD1對急性髓系白血?。ˋML)細(xì)胞增殖、遷移、侵襲和凋亡的影響及其分子機制。
方法 體外培養(yǎng)人骨髓基質(zhì)細(xì)胞系HS-5與AML細(xì)胞系HL-60、THP-1、U-937和Kasumi-1。將THP-1細(xì)胞隨機分為NC組、si-NC組、si-circPCMTD1組、miR-328 NC組、miR-328-3p組和si-circPCMTD1+anti-miR-NC/anti-miR-328-3p組。采用實時熒光定量PCR(RT-qPCR)和Western blot法分別檢測circPCMTD1、miR-328-3p表達量和相關(guān)蛋白的表達量;采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯四唑溴化銨(MTT)法、Transwell實驗和流式細(xì)胞術(shù)分別檢測細(xì)胞增殖、遷移、侵襲和凋亡;并通過雙熒光素酶報告實驗評估circPCMTD1和miR-328-3p的關(guān)系。
結(jié)果 與HS-5細(xì)胞比較,circPCMTD1在AML細(xì)胞中高表達(F=66.258,P<0.05),miR-328-3p則相反(F=101.145,P<0.05)。下調(diào)circPCMTD1抑制了細(xì)胞的增殖、遷移、侵襲及其相關(guān)蛋白表達,并誘導(dǎo)了細(xì)胞的凋亡(F=38.952~290.338,P<0.05)。上調(diào)miR-328-3p可削弱WT-circPCMTD1的熒光素酶活性,且下調(diào)miR-328-3p可減弱circPCMTD1下調(diào)對細(xì)胞增殖的抑制作用(F=42.655,P<0.05)。
結(jié)論 沉默circPCMTD1可通過靶向miR-328-3p抑制AML細(xì)胞的生長。
[關(guān)鍵詞] 白血病,髓樣,急性;RNA,環(huán)狀;微RNAs;細(xì)胞增殖;細(xì)胞運動;腫瘤浸潤;細(xì)胞凋亡
[中圖分類號] R733.72
[文獻標(biāo)志碼] A
[文章編號] 2096-5532(2024)05-0663-06
doi:10.11712/jms.2096-5532.2024.60.162
[網(wǎng)絡(luò)出版] https://link.cnki.net/urlid/37.1517.R.20241111.0913.001;2024-11-11 14:27:38
Effect of circPCMTD1 on malignant behavior of acute myeloid leukemia cells and its mechanism
LU Jingzhong, FANG Yongxiu, HUANG Meiqin, WANG Xiaohong
(Department of Hematology, Chongming Hospital Affiliated to Shanghai University of Health Sciences, Shanghai 202150, China)
[Abstract]Objective To explore the effects of circPCMTD1 on the proliferation, migration, invasion, and apoptosis of acute myeloid leukemia (AML) cells and the underlying molecular mechanism.
Methods Human bone marrow stromal cell line HS-5 and AML cell lines HL-60, THP-1, U-937, and Kasumi-1 were cultured in vitro. THP-1 cells were randomly divided into NC group, si-NC group, si-circPCMTD1 group, miR-328 NC group, miR-328-3p group, si-circPCMTD1+anti-miR-328 NC group, and si-circPCMTD1+anti-miR-328-3p group. Real-time fluorescence quantitative PCR and Western blot were used to mea-
sure the expression of circPCMTD1, miR-328-3p, and related proteins. Cell proliferation, migration, invasion, and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Transwell and flow cytometry methods. The relationship between circPCMTD1 and miR-328-3p was analyzed by dual luciferase reporting assay.
Results Compared with HS-5 cells, circPCMTD1 was highly expressed in AML cells (F=66.258,Plt;0.05), whereas the opposite was true for miR-328-3p (F=101.145,Plt;0.05). Silencing circPCMTD1 inhibited the proliferation, migration, and invasion of cells and the expression of related proteins, and induced cell apoptosis (F=38.952-290.338,Plt;0.05). Upregulation of miR-328-3p impaired the luciferase activity of WT-circPCMTD1, and downregulation of miR-328-3p attenuated the inhibitory effect of circPCMTD1 downregulation on cell proliferation (F=42.655,Plt;0.05).
Conclusion Silencing circPCMTD1 can repress THP-1 cell growth through targeting miR-328-3p.
[Key words] leukemia, myeloid, acute; RNA, circular; microRNAs; cell proliferation; cell movement; neoplasm invasiveness; apoptosis
急性髓系白血?。ˋML)是白血病的4個亞型之一。AML是成人中最常見的急性白血病,發(fā)病率和病死率都很高[1]。盡管化療、支持性治療和造血干細(xì)胞移植等治療方法取得了許多進展,但其對AML病人的療效不理想[2]。因此,深入探索新的治療靶點和闡明疾病病理機制有助于AML的治療。環(huán)狀RNA(circRNA)是一種具有共價閉合環(huán)和高穩(wěn)定性的單鏈RNA分子[3]。有研究結(jié)果表明,circPCMTD1參與調(diào)控人類惡性腫瘤進展[4]。然而,circPCMTD1在白血病中的作用尚不清楚。既往研究表明,miR-328-3p 在AML中的表達明顯減少[5],miR-328-3p低表達與AML病人的不良預(yù)后有關(guān)[6]。雖然miR-328-3p 在AML中的作用已有報道,但 circPCMTD1/miR-328-3p 在AML中的作用尚不清楚。本研究旨在探討circPCMTD1是否能通過靶向 miR-328-3p影響AML細(xì)胞的進展。現(xiàn)將結(jié)果報告如下。
1 材料與方法
1.1 實驗材料
人骨髓基質(zhì)細(xì)胞系HS-5與AML細(xì)胞系HL-60、THP-1、U-937和Kasumi-1購自美國ATCC公司;胎牛血清和RPMI-1640培養(yǎng)液購自美國Gibco公司;Trizol、反轉(zhuǎn)錄和實時熒光定量PCR(RT-qPCR)試劑購自日本Takara公司;3-(4,5-二甲基-2-噻唑基)-2,5二苯基溴化四唑(MTT)和Annexin V-FITC/PI細(xì)胞凋亡檢測試劑盒分別購自上海晶抗生物工程有限公司和上海貝博生物公司;RIPA蛋白裂解液和BCA試劑盒購自北京百奧萊博科技有限公司;Transwell小室和Matrigel基質(zhì)膠購自美國BD公司;雙熒光素酶報告基因檢測試劑盒和Lipofectamine2000分別購自美國Promega公司和Invitrogen公司。
1.2 實驗方法
1.2.1 細(xì)胞轉(zhuǎn)染與分組 THP-1細(xì)胞以80%融合度進行轉(zhuǎn)染?;贚ipofectamine2000的脂質(zhì)體轉(zhuǎn)染法,將si-NC、si-circPCMTD1、miR-328 NC、miR-328-3p分別轉(zhuǎn)染至THP-1細(xì)胞中,分別標(biāo)記為si-NC組、si-circPCMTD1組、miR-328 NC組和miR-328-3p組,而未轉(zhuǎn)染的細(xì)胞標(biāo)記為NC組。另將si-circPCMTD1和anti-miR-328 NC、si-circPCMTD1和anti-miR-328-3p分別共轉(zhuǎn)染至THP-1細(xì)胞中,分別標(biāo)記為si-circPCMTD1+anti-miR-328 NC組和si-circPCMTD1+anti-miR-328-3p組。細(xì)胞轉(zhuǎn)染48 h后,用于后續(xù)實驗。
1.2.2 RT-qPCR檢測各組circPCMTD1和miR-328-3p表達 先提取細(xì)胞總RNA并反轉(zhuǎn)錄合成cDNA,再按RT-qPCR試劑盒說明書步驟行PCR擴增,最后采用2-△△Ct方法計算各組circPCMTD1和miR-328-3p相對于GAPDH和U6的表達水平。circPCMTD1、miR-328-3p、GAPDH、U6的引物由生工生物工程公司合成。
1.2.3 MTT法檢測細(xì)胞活性 依據(jù)文獻報道的方法進行細(xì)胞活性檢測[7]。在酶標(biāo)儀450 nm波長處測定THP-1細(xì)胞(2.5×107/L)的吸光度。
1.2.4 Transwell侵襲實驗檢測細(xì)胞遷移及侵襲能力 依據(jù)文獻報道的方法進行Transwell實驗[8]。下室表面遷移或侵襲的細(xì)胞經(jīng)固定和染色后,在顯微鏡下觀察并分析隨機選取的5個視野內(nèi)的遷移或侵襲細(xì)胞數(shù)。
1.2.5 流式細(xì)胞術(shù)檢測細(xì)胞凋亡 依據(jù)文獻報道的方法進行流式細(xì)胞術(shù)檢測[9]。收集的細(xì)胞經(jīng)PBS漂洗后,添加結(jié)合緩沖液對細(xì)胞進行重懸。 隨后,將5 μL Annexin V-FITC摻入細(xì)胞內(nèi),置于5 μL碘化丙啶(PI)中15 min后檢測。用流式細(xì)胞儀檢測細(xì)胞的凋亡率。
1.2.6 Western blot法檢測細(xì)胞中蛋白的表達 依據(jù)文獻報道的方法檢測各組細(xì)胞增殖、遷移、侵襲和凋亡相關(guān)蛋白(包括PCNA、MMP-2、MMP-9、Bcl-2和Bax)的表達水平[10]。提取的細(xì)胞總蛋白經(jīng)SDS-PAGE分離后轉(zhuǎn)膜,封閉后,加一抗4 ℃孵育過夜,隨后浸泡在二抗稀釋液中室溫孵育2 h。曝光顯影,應(yīng)用Image J軟件分析條帶灰度值。
1.2.7 雙熒光素酶報告基因?qū)嶒烌炞CcircPCMTD1和miR-328-3p的作用關(guān)系 將WT-circPCMTD1(野生型)和MUT-circPCMTD1(突變型)分別與miR-328 NC和miR-328-3p共轉(zhuǎn)染至THP-1細(xì)胞中,按照說明分析細(xì)胞的熒光素酶活性。
1.3 統(tǒng)計學(xué)處理
采用SPSS 21.0軟件進行統(tǒng)計學(xué)處理。計量資料數(shù)據(jù)以±s表示,兩組或多組比較采用獨立樣本t檢驗和單因素方差分析。以P<0.05為差異有統(tǒng)計學(xué)意義。
2 結(jié) "果
2.1 AML細(xì)胞中circPCMTD1和miR-328-3p的表達
各組細(xì)胞circPCMTD1和miR-328-3p表達比較,差異均有統(tǒng)計學(xué)意義(F=66.258、101.145,P<0.05)。與HS-5細(xì)胞相比較,AML細(xì)胞系HL-60、THP-1、U-937、Kasumi-1細(xì)胞circPCMTD1表達明顯升高(P<0.05),而miR-328-3p表達明顯降低(P<0.05)。見表1。
2.2 敲低circPCMTD1對THP-1細(xì)胞惡性行為的影響
各組circPCMTD1表達、細(xì)胞活力、遷移和侵襲細(xì)胞數(shù)目、凋亡率、相關(guān)蛋白表達比較差異均有統(tǒng)計學(xué)意義(F=38.952~290.338,P<0.05)。與si-NC組比較,si-circPCMTD1組的circPCMTD1表達、細(xì)胞活力、遷移和侵襲細(xì)胞數(shù)目以及PCNA、MMP-2、MMP-9、Bcl-2蛋白表達明顯降低(P<0.05),而凋亡率和Bax表達明顯增加(P<0.05)。見圖1、表2。
2.3 circPCMTD1和miR-328-3p的作用關(guān)系
Starbase分析發(fā)現(xiàn)circPCMTD1的一段核苷酸序列與miR-328-3p結(jié)合。見圖2。上調(diào)miR-328-3p降低了WT-circPCMTD1的熒光素酶活性(t=17.830,P<0.05)。見表3。敲減(si-circPCMTD1)和過表達circPCMTD1(pcDNA-circPCMTD1)分別上調(diào)和下調(diào)miR-328-3p,即circPCMTD1負(fù)調(diào)控miR-328-3p的表達(t=11.550~17.190,P<0.05)。見表4。
2.4 上調(diào)miR-328-3p對THP-1細(xì)胞惡性行為的影響
各組miR-328-3p表達、細(xì)胞活力、遷移和侵襲細(xì)胞數(shù)目、凋亡率、相關(guān)蛋白表達比較差異均有統(tǒng)計
學(xué)意義(F=34.020~466.200,P<0.05)。與miR-NC組比較,miR-328-3p組miR-328-3p表達、凋亡率以及Bax蛋白表達顯著增加(P<0.05),而細(xì)胞活力、遷移和侵襲細(xì)胞數(shù)目以及PCNA、MMP-2、MMP-9、Bcl-2蛋白表達水平明顯下降(P<0.05)。見圖3、表5。
2.5 敲低miR-328-3p對下調(diào)circPCMTD1介導(dǎo)的THP-1細(xì)胞生長抑制作用影響
與si-circPCMTD1+anti-miR-328 NC組比較,si-circPCMTD1+anti-miR-328-3p組的miR-328-3p表達、凋亡率以及Bax蛋白表達明顯降低(F=56.720~87.840,P<0.05),而細(xì)胞活力、遷移和侵襲細(xì)胞數(shù)目以及PCNA、MMP-2、MMP-9、Bcl-2蛋白表達水平明顯升高(F=42.655~200.800,P<0.05)。見圖4、表6。
3 討 "論
越來越多的證據(jù)表明,circRNA參與了白血病的發(fā)生和發(fā)展。circ_0000143在T細(xì)胞急性淋巴細(xì)胞白血病中的表達降低,其過表達可抑制T細(xì)胞急性淋巴細(xì)胞白血病細(xì)胞的存活、遷移和侵襲,并誘導(dǎo)細(xì)胞凋亡[11]。過表達circ_0000745可促進T細(xì)胞急性淋巴細(xì)胞白血病細(xì)胞增殖并抑制其凋亡,抑制其表達則結(jié)果相反[12]。下調(diào)circ_0009910可抑制AML細(xì)胞的球體形成和自噬,并促進其凋亡[13]。circ_0012152在AML組織和細(xì)胞中表達增加,敲減circ_0012152可抑制AML細(xì)胞增殖,并促進細(xì)胞凋亡[14]。circ_0002483在AML中表達升高,其表達下調(diào)可抑制AML細(xì)胞增殖,促進細(xì)胞周期阻滯和細(xì)胞凋亡[15]。本文結(jié)果顯示,circPCMTD1在AML細(xì)胞中表達上調(diào),其表達下調(diào)可以抑制細(xì)胞增殖、遷移和侵襲,并增加細(xì)胞的凋亡率。提示敲低circPCMTD1可削弱THP-1細(xì)胞的生長。
既往研究表明,circPCMTD1可以作為競爭性內(nèi)源RNA抑制miRNA,從而參與促進膠質(zhì)瘤進展[4]。本研究Starbase分析發(fā)現(xiàn),circPCMTD1的一段核苷酸序列與miR-328-3p互補。同時,miR-328-3p上調(diào)可降低WT-circPCMTD1熒光素酶活性,且上調(diào)和下調(diào)circPCMTD1分別顯著降低和升高miR-328-3p的表達,即circPCMTD1靶向負(fù)調(diào)控miR-328-3p表達。大量研究表明,miR-328-3p可抑制多種癌細(xì)胞的惡性行為。例如,上調(diào)miR-328-3p
表達可抑制乳癌細(xì)胞的生長和轉(zhuǎn)移[16]。miR-328-3p模擬物可抑制肺腺癌細(xì)胞的增殖和遷移,促進細(xì)胞凋亡[17]。上調(diào)miR-328-3p減弱了結(jié)直腸癌細(xì)胞的生長和轉(zhuǎn)移[18]。miR-328-3p可通過調(diào)控整合素α5限制膀胱癌細(xì)胞的生長,其低表達可預(yù)示膀胱癌病人預(yù)后不良[19]。本文研究結(jié)果顯示,miR-328-3p在AML細(xì)胞中表達下調(diào),而其上調(diào)削弱了THP-1細(xì)胞的生長和轉(zhuǎn)移能力。提示miR-328-3p限制了THP-1細(xì)胞的惡性行為;此外,miR-328-3p的下調(diào)減弱了沉默circPCMTD1對THP-10細(xì)胞生長和轉(zhuǎn)移的抑制作用。提示circPCMTD1可通過靶向miR-328-3p影響THP-1細(xì)胞的惡性行為。
綜上所述,circPCMTD1在AML細(xì)胞中表達上調(diào),其敲低可以通過靶向調(diào)控miR-328-3p抑制THP-1細(xì)胞的惡性表型,這為治療AML提供了新的潛在靶點。然而,本研究的不足之處為僅限于體外實驗,circPCMTD1在體內(nèi)的作用和具體的調(diào)控機制有待進一步探討。
[參考文獻]
[1]PRADA-ARISMENDY J, ARROYAVE J C, RTHLIS-
BERGER S. Molecular biomarkers in acute myeloid leukemia[J]. Blood Reviews, 2017,31(1):63-76.
[2]TIAN M, GONG W J, GUO J M. Long non-coding RNA SNHG1 indicates poor prognosis and facilitates disease progression in acute myeloid leukemia[J]. Biology Open, 2019,8(10):bio046417.
[3]ZHANG Z K, XIE Q, HE D M, et al. Circular RNA: new star, new hope in cancer[J]. BMC Cancer, 2018,18(1):834.
[4]ZHENG S Q, QI Y, WU J, et al. CircPCMTD1 acts as the sponge of miR-224-5p to promote glioma progression[J]. Frontiers in Oncology, 2019,9:398.
[5]XUE L, LI C H, REN J, et al. KDM4C contributes to cytarabine resistance in acute myeloid leukemia via regulating the miR-328-3p/CCND2 axis through MALAT1[J]. Therapeutic Advances in Chronic Disease, 2021,12:2040622321997259.
[6]LIU L, CHEN R A, ZHANG Y P, et al. Low expression of circulating microRNA-328 is associated with poor prognosis in patients with acute myeloid leukemia[J]. Diagnostic Pathology, 2015,10:109.
[7]HUANG L F, HUANG L, MING X, et al. CircNFIX knockdown inhibited AML tumorigenicity by the miR-876-3p/TRIM31 axis[J]. Hematology, 2022, 27(1):1046-1055.
[8]ZHANG T, ZHOU Y, GUAN J, et al. Circ_0058058 knockdown inhibits acute myeloid leukemia progression by sponging miR-4319 to regulate EIF5A2 expression[J]. Cancer Biotherapy amp; Radiopharmaceuticals, 2023,38(10):738-748.
[9]WANG J, WU C P, ZHOU W L. CircPLXNB2 regulates acute myeloid leukemia progression through miR-654-3p/CCND1 axis[J]. Hematology (Amsterdam, Netherlands), 2023, 28(1):2220522.
[10]WANG Y Y, GUO T, LIU Q, et al. CircRAD18 accelerates the progression of acute myeloid leukemia by modulation of miR-206/PRKACB axis[J]. Cancer Management and Research, 2020,12:10887-10896.
[11]TONG J, LIU H L, YAO W, et al. Withdrawal: Down-regulation of circRNA_0000143 regulates miR-142-3p/GRα axis to facilitate the progression of T-ALL[J]. Cancer Science, 2023,114(4):1776.
[12]FENG H H, LI F, TANG P. Circ_0000745 regulates NOTCH1-mediated cell proliferation and apoptosis in pediatric T-cell acute lymphoblastic leukemia through adsorbing miR-193b-3p[J]. Hematology (Amsterdam, Netherlands), 2021, 26(1):885-895.
[13]WU Y W, ZHAO B, CHEN X H, et al. Circ_0009910 sponges miR-491-5p to promote acute myeloid leukemia progression through modulating B4GALT5 expression and PI3K/AKT signaling pathway[J]. International Journal of Laboratory Hematology, 2022,44(2):320-332.
[14]SHANG Z, MING X, WU J Y, et al. Downregulation of circ_0012152 inhibits proliferation and induces apoptosis in acute myeloid leukemia cells through the miR-625-5p/SOX12 axis[J]. Hematological Oncology, 2021,39(4):539-548.
[15]XIAO Y, MING X, WU J Y. Hsa_circ_0002483 regulates miR-758-3p/MYC axis to promote acute myeloid leukemia progression[J]. Hematological Oncology, 2021,39(2):243-253.
[16]LI J, LI Y M, CHENG H. Circ-RPPH1 knockdown retards breast cancer progression via miR-328-3p-mediated suppression of HMGA2[J]. Clinical Breast Cancer, 2022, 22(3):e286-e295.
[17]LU J C, LIN J H, ZHOU Y, et al. MiR-328-3p inhibits lung adenocarcinoma-genesis by downregulation PYCR1[J]. Biochemical and Biophysical Research Communications, 2021,550:99-106.
[18]PAN S, REN F, LI L, et al. MiR-328-3p inhibits cell prolife-
ration and metastasis in colorectal cancer by targeting Girdin and inhibiting the PI3K/Akt signaling pathway[J]. Experimental Cell Research, 2020,390(1):111939.
[19]YAN T, YE X X. MicroRNA-328-3p inhibits the tumorigenesis of bladder cancer through targeting ITGA5 and inactivating PI3K/AKT pathway[J]. European Review for Medical and Pharmacological Sciences, 2019, 23(12):5139-5148.
(本文編輯 牛兆山)
[收稿日期]2024-05-15; [修訂日期]2024-07-15
[基金項目]上海市崇明區(qū)“可持續(xù)發(fā)展科技創(chuàng)新行動計劃”項目(CKY2022-04)
[第一作者]陸靜忠(1980-),男,碩士,主治醫(yī)師。
[通信作者]王小紅(1980-),女,統(tǒng)計師。E-mail:ysfwxh502@163.com。