Regulation of the P2X7R by microRNA-216b in human breast cancer

Breast cancer is the most common cancer in women around the world. However, the molecular mechanisms underlying breast cancer pathogenesis are only partially understood. Here, in this study, we found that P2X7R was up-regulated and miR-216b was down-regulated in breast cancer cell lines and tissues. Using bioinformatic analysis and 3′UTR luciferase reporter assay, we determined P2X7R can be directly targeted by miR-216b, which can down-regulate endogenous P2X7R mRNA and protein levels. Ectopic expression of miR-216b mimics leads to inhibited cell growth and apoptosis, while blocking expression of the miR-216b results in increased cell proliferation. Furthermore, our findings demonstrate that knockdown of P2X7R promotes apoptosis in breast cancer cells through down-regulating Bcl-2 and increasing the cleavage caspase-3 protein level. Finally, we confirmed that down-regulation of miR-216b in breast cancer is inversely associated with P2X7R expression level. Together, these findings establish miR-216b as a novel regulator of P2X7R and a potential therapeutic target for breast cancer.     

MiR-206与ANXA2的3′UTR靶向结合抑制乳腺癌侵袭

膜联蛋白A2（annexin A2,ANXA2）可促进人结直肠癌的侵袭和迁移。然而,ANXA2在乳腺癌中的作用以及调节机制尚缺乏系统的研究。本研究旨在探讨微小RNA-206（microRNA-206,miR-206）如何调节ANXA2基因的表达,进而影响乳腺癌的侵袭。通过基因预测软件TargetScan (TargetScan V5.2)找到与ANXA2的3′UTR区互补结合的miR-206。运用实时定量 PCR（qRT-PCR）检测不同乳腺癌细胞系中miR-206的表达水平,发现低侵袭性乳腺癌MCF-7细胞株miR-206 表达量明显高于高侵袭性乳腺癌细胞株MDA-231、MDA-435和T47D。运用转染技术将 miR-206 质粒及miR-206 抑制剂转入乳腺癌细胞系MDA-231后,qRT-PCR检测转染后各组细胞中miR-206的表达情况,结果显示转染成功。用Western印迹法检测各组细胞中ANXA2的表达情况,结果显示,miR-206负向调控ANXA2蛋白的表达。 qRT-PCR显示,过表达乳腺癌细胞内miR-206 后,ANXA2 mRNA基本没有变化。结果显示,miR-206是在翻译水平上影响ANXA2蛋白的表达。荧光素酶实验显示：miR-206能特异性地与ANXA2 mRNA的3′UTR结合,抑制其荧光素酶活性。Transwell侵袭实验检测各组细胞的侵袭能力。结果显示,过表达miR-206后,乳腺癌细胞体外侵袭能力明显减弱。综上所述,miR-206 通过靶向结合癌基因ANXA2 mRNA的3′UTR区,抑制ANXA2蛋白翻译,从而抑制了乳腺癌细胞的侵袭。因此,miR-206有望成为抑制乳腺癌侵袭与治疗乳腺癌的新靶点和生物学标记物。     

miR-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为研究

目的:探讨mi R-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为。方法:qRT-PCR法检测并比较肺癌组织、癌旁正常组织、肺癌细胞、正常肺上皮细胞中的mi R-199a-3p m RNA相对表达量。比较远处转移肺癌组织、未转移肺癌组织中mi R-199a-3p m RNA相对表达量。qRT-PCR法、Western Blot法检测并比较肺癌组织、癌旁正常组织中的CBX7 m RNA及蛋白的表达水平。荧光素酶活性法检测mi R-199a-3p与靶基因CBX7的结合。比较mi R-199a-3p模拟物转染组与阴性对照组的肺癌细胞中的CBX7 m RNA相对表达量及CBX7蛋白表达水平。CCK8实验检测mi R-199a-3p对肺癌细胞增殖的促进作用。Tranwell实验检测mi R-199a-3p对肺癌细胞侵袭与迁移能力的影响。结果:肺癌组织中mi R-199a-3p明显高于癌旁正常组织,发生远处转移的肺癌组织中mi R-199a-3p m RNA的表达量明显高于未发生转移的肺癌组织,差异有统计学意义(P<0.001)。肺癌组织中CBX7m RNA、CBX7蛋白表达水平均明显低于癌旁正常组织,差异有统计学意义(P<0.001)。荧光素酶活性法证实mi R-199a-3p可与靶基因CBX7结合抑制CBX7的表达。肺癌细胞中mi R-199a-3p m RNA的相对表达量明显高于正常肺上皮细胞,CBX7 m RNA相对表达量明显低于正常肺上皮细胞(P<0.05)。对于肺癌细胞,mi R-199a-3p模拟物转染组的CBX7 m RNA相对表达量及CBX7蛋白表达水平均明显低于阴性对照组(P<0.001)。CCK8实验证实mi R-199a-3p能够促进肺癌细胞的增殖,Tranwell实验证实mi R-199a-3p对肺癌细胞侵袭与迁移具有积极的促进作用。结论:mi R-199a-3p在肺癌的发生发展过程中发挥重要作用,能够通过抑制CBX7基因的表达,促进肺癌细胞的增殖、侵袭和转移。     

miR-223 regulates migration and invasion by targeting Artemin in human esophageal carcinoma

Background

Artemin (ARTN) is a neurotrophic factor belonging to the glial cell-derived neurotrophic factor family of ligands. To develop potential therapy targeting ARTN, we studied the roles of miR-223 in the migration and invasion of human esophageal carcinoma.

Methods

ARTN expression levels were detected in esophageal carcinoma cell lines KYSE-150, KYSE-510, EC-9706, TE13, esophageal cancer tissues and paired non-cancerous tissues by Western blot. Artemin siRNA expression vectors were constructed to knockdown of artemin expression mitigated migration and invasiveness in KYSE150 cells. Monolayer wound healing assay and Transwell invasion assay were applied to observe cancer cell migration and invasion. The relative levels of expression were quantified by real-time quantitative PCR.

Results

ARTN expression levels were higher in esophageal carcinoma tissue than in the adjacent tissue and was differentially expressed in various esophageal carcinoma cell lines. ARTN mRNA contains a binding site for miR-223 in the 3'UTR. Co-transfection of a mir-223 expression vector with pMIR-ARTN led to the reduced activity of luciferase in a dual-luciferase reporter gene assay, suggesting that ARTN is a target gene of miR-223. Overexpression of miR-223 decreased expression of ARTN in KYSE150 cells while silencing miR-223 increased expression of ARTN in EC9706 cells. Furthermore, overexpression of miR-223 in KYSE150 cells decreased cell migration and invasion. Silencing of miR-223 in EC9706 cells increased cell migration and invasiveness.

Conclusions

These results reveal that ARTN, a known tumor metastasis-related gene, is a direct target of miR-223 and that miR-223 may have a tumor suppressor function in esophageal carcinoma and could be used in anticancer therapies.     

miR-144 reverses cisplatin resistance in cervical cancer via targeting LHX2

Mounting evidence showed that microRNAs involve in development and chemoresistance of various human cancers. We explored the roles and mechanisms of miR-144 in resistance to cisplatin (CDDP) of cervical cancer cells. miR-144 and LIM homeobox 2 (LHX2) expression in CDDP-resistant and the parental cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. The functions of miR-144 overexpression on cell viability, the incidence of apoptosis, the activity of caspase-3/7, the cleaved-caspase-3 expression, cell migration, and invasion were determined in Hela cells and Hela/CDDP cells. Overexpression of miR-144 reduced cell viability, induced cell apoptosis, and inhibited cell migration and invasion after CDDP treatment. Besides, a luciferase reporter system demonstrated that miR-144 could directly bind to the 3′ untranslated region (3′-UTR) of LHX2 messenger RNA (mRNA). Gain expression of miR-144 decreased the expression of LHX2 both in mRNA and protein levels. Furthermore, restoration of LHX2 partly abolished the biological functions of miR-144 in resistance of cervical cancer cells. Taken together, miR-144 overcomes resistance to CDDP via promoting cell apoptosis and inhibiting invasion through targeting LHX2 in cervical cancer cells.     

miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1对卵巢癌细胞增殖的影响

目的: 探讨miR-335 靶向Rho相关卷曲螺旋形成蛋白激酶1(rho associated coiled-coil forming protein kinase 1,ROCK1)对卵巢癌细胞系SKOV3增殖的调控作用。方法:(1)选取卵巢癌细胞系SKOV3及人正常卵巢上皮细胞系IOSE80,采用RT-PCR检测各组细胞中miR-335表达;采用Western blot检测各组细胞中ROCK1蛋白表达;(2)选取卵巢癌细胞系SKOV3,分别转染miR-335 mimic及mimic control,采用RT-PCR检测细胞中miR-335表达;(3)选取卵巢癌细胞系SKOV3,将SKOV3荧光素酶报告载体与miR-335 mimic共转染,采用荧光素酶活性实验验证miR-335对SKOV3的靶向作用;(4)选取卵巢癌细胞系SKOV3,分为3组,即SKOV3组(转染mimic control)、miR-335 mimic组(转染miR-335 mimic)及miR-335 mimic+ROCK1组(共转染miR-335 mimic+ROCK1),采用MTT法检测各组细胞增殖活性,采用Western blot检测各组细胞中ROCK1蛋白表达,采用RT-PCR检测细胞中Cyclin D1表达。结果: (1)RT-PCR结果显示,卵巢癌细胞SKOV3中miR-335表达显著低于人正常卵巢上皮细胞IOSE80(P < 0.05);Western blot结果显示,卵巢癌细胞SKOV3中ROCK1蛋白表达显著高于人正常卵巢上皮细胞IOSE80(P < 0.05);(2)RT-PCR结果显示,转染miR-335 mimic可使卵巢癌细胞SKOV3中miR-335表达上调,与转染mimic control相比较差异具有统计学意义(P < 0.05);(3)双荧光素酶活性检测结果显示,miR-335 mimic可显著抑制野生型ROCK1-Wt报告载体的荧光素酶活性,但对突变型ROCK1-Mut报告载体的荧光素酶活性并无显著抑制作用;(4)转染miR-335mimic后,卵巢癌细胞SKOV3增殖活性及Cyclin D1表达较阴性对照组显著降低(P < 0.05);而转染miR-335 mimic+ROCK1后,卵巢癌细胞SKOV3增殖活性及Cyclin D1表达较单纯转染miR-335 mimic组显著提高(P < 0.05),但仍显著低于阴性对照组(P < 0.05)。Western blot检测结果显示,转染miR-335mimic后,卵巢癌细胞SKOV3中ROCK1蛋白表达较阴性对照组显著降低(P < 0.05);而转染miR-335 mimic+ROCK1后,ROCK1蛋白表达较单纯转染miR-335mimic组显著增高(P < 0.05),且显著高于阴性对照组(P < 0.05)。结论: miR-335可通过靶向ROCK1抑制卵巢癌细胞系SKOV3增殖。     

MiR-150-5p通过靶向调控Rab1A抑制乳腺癌细胞MDA-231的上皮-间质转化

先前的研究表明,miR-150-5p发挥抑癌基因的作用,调控肿瘤细胞的侵袭与转移。然而,关于其在乳腺癌细胞侵袭与转移中的机制尚不明确。本实验旨在研究miR-150-5p负向调控Rab1A在乳腺癌细胞上皮-间质转化（epithelial-mesenchymal transition,EMT）中的作用。双荧光素酶的结果显示,miR-150-5p可负向调控Rab1A。荧光定量PCR (qRT-PCR) 结果显示,miR-150-5p在乳腺癌细胞MCF-7及MDA-MB-231（MDA-231）中的表达水平明显低于正常乳腺上皮细胞MCF-10A; 在MDA-231中过表达miR-150-5p后,qRT-PCR结果显示,Rab1A mRNA的表达水平明显降低。Western印迹结果显示,过表达miR-150-5p后,miR-150-5p组细胞中的Rab1A、波形蛋白(vimentin)及N-钙黏着蛋白(N-cadherin)的表达水平相对于对照组（NC）细胞明显降低,而E-钙黏着蛋白(E-cadherin)的表达水平明显增加。Transwell侵袭和划痕实验显示,与miR-150-5p+Con组细胞相比,miR-150-5p+Rab1A组细胞的侵袭和迁移能力明显增加。qRT-PCR结果显示,miR-150-5p+Rab1A组细胞的Rab1A mRNA表达水平明显增加。Western印迹结果显示,miR-150-5p+Rab1A组细胞中的波形蛋白、N-钙黏着蛋白表达水平明显增加, 而E-钙黏着蛋白表达明显降低,过表达Rab1A后显著逆转了miR-150-5p对EMT的影响。综上所述,miR-150-5p可以通过负向调控Rab1A抑制EMT,进而抑制乳腺癌细胞的侵袭和迁移。     

miR-613 inhibits cell migration and invasion by downregulating Daam1 in triple-negative breast cancer

Dishevelled-associated activator of morphogenesis 1 (Daam1) is a formin protein and participates in regulating cell migration of triple-negative breast cancer (TNBC) cells. The specific miRNA targeting Daam1 and mediating cell migration and invasion remains obscure. This experiment investigated the suppressive role of miR-613 in TNBC cells. The luciferase activity of Daam1 3′-untranslated region (3′-UTR) based reporters constructed in HEK-293T and MCF-7 cells suggested that Daam1 was the target gene of miR-613. Overexpressed miR-613 reduced the protein level of Daam1, weakened RhoA activity, and retarded the cell migration, cell invasion and colony formation of TNBC cells. Overexpression of Daam1 or RhoA rescued cell migration and invasion in miR-613-overexpressed TNBC cells, but failed to reverse colony formation. MiR-613 was significantly downregulated in breast cancer tissues compared with that in adjacent normal tissues. This downregulation in TNBC tissues and lymphnode metastatic breast cancer tissues was more obvious than that in non-TNBC tissues and non-metastatic cancer tissues, respectively. MiR-613 weakens the resistance of TNBC cells against paclitaxel rather than adriamycin, cyclophosphamide, docetaxel, and kaempferol. Taken together, miR-613 is involved in cell migration and invasion of TNBC cells via targeting Daam1/RhoA signaling pathway.     

miR-29a靶向调控PDGFB促进非小细胞肺癌细胞凋亡

为了探究miR-29a对非小细胞肺癌细胞增殖和凋亡的影响及分子机制,本研究通过荧光定量PCR检测肺癌组织、癌旁组织、肺癌细胞以及人正常肺支气管上皮细胞BEAS-2B中miR-29a的表达,在肺癌A549转染miR-29a mimics后,使用荧光定量PCR和CCK-8法分别检测miR-29a的表达以及各组细胞的活力,使用流式细胞术检测A549细胞凋亡;通过荧光定量PCR检测肺癌组织、癌旁组织PDGFB m RNA的表达,采用Western blot检测PDGFB蛋白的表达;使用双荧光素酶报告基因检测miR-29a可能的靶基因;在肺癌A549细胞转染miR-29a mimics后继续转染PDGFB过表达质粒,通过qPCR和Western blotting分别检测PDGFB mRNA和蛋白的表达。结果表明,与癌旁组织相比,miR-29a在肺癌组织的表达显著下调(p<0.01),PDGFB在肺癌组织的表达显著增加(p<0.01);转染miR-29a mimics后,肺癌A549细胞中miR-29a表达显著增加(p<0.01);CCK-8法结果显示miR-29a mimics组A549肺癌细胞在24 h和48 h后细胞增值率较miR-NC对照组显著降低(p<0.01);流式细胞术结果显示miR-29a mimics组的细胞凋亡率较miR-NC对照组显著增加(p<0.01);与miR-NC+PDGFB 3’UTR WT组相比,miR-29a mimics+PDGFB 3’UTR WT组的荧光强度显著降低(p<0.01);荧光定量PCR和Western blotting显示miR-29a mimics+PDGFB组PDGFB m RNA和蛋白表达量与miR-29a mimics+vector组相比显著增加(p<0.01)。本研究结果表明miR-29a在肺癌组织和肺癌细胞株中低表达,及抑制PDGFB的表达并且促进肺癌细胞凋亡。     

c-Myb is an evolutionary conserved miR-150 target and miR-150/c-Myb interaction is important for embryonic development

Human c-Myb proto-oncogene is highly expressed in hematopoietic progenitors as well as leukemia and certain solid tumor. However, the regulatory mechanisms of its expression and biological functions remain largely unclear. Recently, c-Myb has been shown to be targeted by microRNA-150 (miR-150) which thereby controls B cell differentiation in mice. In this study, we demonstrated that c-Myb is an evolutionary conserved target of miR-150 in human and zebrafish, using reporter assays. Ectopic expression of miR-150 in breast cancer and leukemic cells repressed endogenous c-Myb at both messenger RNA (mRNA) and protein levels. Among several leukemia cell lines, primary leukemia cells, and normal lymphocytes, expression levels of miR-150 inversely correlated with c-Myb. The miR-150 overexpression or c-Myb silencing in zebrafish zygotes led to similar and serious phenotypic defects in zebrafish, and the phenotypic aberrations induced by miR-150 could be reversed by coinjection of c-Myb mRNA. Our findings suggest that c-Myb is an evolutionally conserved target of miR-150 and miR-150/c-Myb interaction is important for embryonic development and possibly oncogenesis.     

miR-125a-5p通过靶向调控GAB2抑制乳腺癌细胞的迁移能力

miR-125a-5p可负性调节GAB2表达,抑制胶质瘤细胞的侵袭和转移。本研究旨在证明miR-125a-5p抑癌作用的普遍性,即miR-125a-5p是否可通过靶向抑制GAB2抑制乳腺癌细胞的迁移。荧光素酶实验结果显示,miR-125a-5p可特异识别GAB2的3′-UTR,抑制报告酶的表达。荧光定量PCR结果揭示,与正常乳腺上皮细胞MCF-10A比较,miR-125a-5p在乳腺癌细胞MDA231和MCF-7中的表达明显降低;与迁移能力相对较低的MCF-7细胞比较,miR-125a-5p在迁移能力较高的MDA231细胞中的表达量更低。Western 印迹结果证明,与空载体（对照）和anti-miR125a 5p转染细胞比较,转染miR-125a-5p明显抑制GAB2蛋白在乳腺癌细胞中的表达。Transwell结果显示,与空载体转染的对照细胞比较,转染miR-125a-5p的乳腺癌细胞穿过基质胶的细胞数明显减少;相反,转染anti-miR125a-5p的细胞穿过基质胶的细胞数却明显增多。上述结果提示,miR-125a-5p在正常的乳腺细胞中高表达,而在乳腺癌细胞中低表达,其表达水平与癌细胞的迁移能力和GAB2表达呈反向关系。本研究结果还提示,miR-125a-5p通过靶向负调控GAB2抑制乳腺癌细胞的迁移能力。总之,本研究证明,miR-125a-5p在肿瘤中发挥抑癌作用。     

miR-19a targets CLCA4 to regulate the proliferation,migration, and invasion of colorectal cancer cells

The role of miR-19a in colorectal cancer (CRC), a devastating disease with high mortality and morbidity, remains controversial. In the present study, we show that the level of miR-19a is significantly higher in clinical CRC tissue samples than in paracancerous tissue samples, and significantly higher in CRC cells lines HT29, SW480, and CaCO2 than in the normal human colon mucosal epithelial cell line NCM460. miR-19a mimics and inhibitors were synthesized and validated. Overexpression of miR-19a mimics significantly promoted, while miR-19a inhibitors inhibited, the proliferation, survival, migration, and invasion of SW480 and CaCO2 CRC cells. Furthermore, mRNA and protein levels of chloride channel accessory 4 (CLCA4) were lower in CRC cells and tissues. Bioinformatics and a luciferase reporter assay confirmed that CLCA4 was a miR-19a target. Further, miR-19a inhibition increased CLCA4 expression. The inhibitory effect of miR-19a on cell growth, survival, migration, and invasion was reversed by knockdown of CLCA4 expression. The data demonstrated that the miR-19a/CLCA4 axis modulates phospho-activation of the PI3K/AKT pathway in CRC cells. In conclusion, our results revealed that miR-19a overexpression decreases CLCA4 levels to promote CRC oncogenesis, suggesting that miR-19a inhibitors have potential applications for future therapeutic of CRC.Key words: Colorectal cancer, miR-19a, CLCA4, proliferation, migration, invasion     

miR-129-5p调节小鼠乳腺上皮细胞内Igf-1的表达

MicroRNAs(miRNAs)是一类大约22个核苷酸的非编码RNA.它能通过调控生长因子表达,引发肿瘤形成、细胞增殖和组织器官发育.本文通过构建miR-129-5p靶基因序列的双荧光素酶报告载体分析了miR-129-5p与靶基因之间的关系,应用脂质体转染技术和实时荧光定量技术观察了miR-129-5p在小鼠乳腺上皮细胞中的表达量及其变化,通过CASY细胞活力仪检测转染后的细胞增殖与活力变化,采用Western 印迹方法检Igf-1的变化.结果表明：miR-129-5p在小鼠乳腺青春期表达最高,成功构建了Igf-1基因 3′UTR荧光素酶报告载体, miR-129-5p抑制其荧光素酶活性（P <0.01）,转染抑制子后miR-129-5p表达降低（P < 0.01）,胰岛素样生长因子（Igf-1)表达增强（P <0.05）,细胞增殖和活力增强（P <0.01）,结果提示：miR-129-5p可能通过抑制靶基因蛋白Igf 1的表达,进而抑制小鼠乳腺上皮细胞增殖和活力.     

鸡miR-19a、miR-19b通过靶向抑制LIN9促进前脂肪细胞增殖

miR-17-92基因簇编码包括miR-19a、miR-19b在内的至少6个miRNA,在鸡细胞的增殖、分化、凋亡及发育等多种生物学过程中发挥重要作用,但其作用机制尚不清楚。生物信息学分析显示,细胞周期调控子LIN9是 miR-17-92 基因簇成员miR-19a和miR-19b的潜在靶点。为验证这一预测,构建含野生型LIN9 3′-UTR荧光素酶报告基因载体（psi-CHECK2-LIN9-3′-UTR-WT）及突变型报告基因载体（psi-CHECK2-LIN9-3′-UTR-MUT）,开展靶标LIN9的鉴定。复合转染结合报告基因酶活性测定结果表明,过表达miR-19a和miR-19b能显著抑制含野生型LIN9 3′-UTR的报告基因表达,而过表达miR-19a和miR-19b抑制剂显著提高野生型LIN9 3′-UTR报告基因的表达。实时定量PCR（RT-qPCR）证明,miR-19a和miR-19b 抑制剂对内源性LIN9 mRNA的表达没有影响,提示miR-19a和miR-19b可能不是通过降解mRNA调控LIN9表达。转染结合CCK-8细胞增殖分析显示,在鸡前脂肪细胞过表达LIN9 明显抑制细胞增殖。与此相一致的是,细胞增殖标志分子CyclinD1、c-Myc、PCNA、Ki67 的mRNA表达量明显降低。本研究证实,LIN9是miR-17-92 基因簇成员miR-19a和miR-19b的靶标,同时证实,LIN9抑制鸡前脂肪细胞的增殖。是否miR-17-92基因簇编码的其它mi-RNA成员也以LIN9为靶标尚不得而知,我室正在研究中。     

Effects of miR-21 downregulation and silibinin treatment in breast cancer cell lines

Silibinin is a natural polyphenol with high antioxidant and anticancer properties, which causes cell cycle arrest and apoptosis in most cancer cell types including breast cancer, but the in-line mechanisms, are still unknown. Silibinin significantly downregulated oncomiR miR-21 expression in breast cancer cells. Here the effect of anti-miR-21 on cell viability, apoptotic induction, cell cycle distribution, and the expression levels of downstream targets of miR-21 were investigated in MCF-7 and T47D cells. MiR-21 mimic transfection was also applied in silibinin treated samples to evaluate functional role of miR-21downregulation on silibinin effects. It was found that after anti-miR-21 transfection, no significant changes were detected in cell viability, apoptosis (except early apoptosis), and cell cycle in MCF-7 and T47D cells. Compared to silibinin, miR-21 mimic transfection in combination with silibinin caused a slight modulation in some of the examined silibinin effects including apoptosis, Bcl2 mRNA and PTEN mRNA and protein levels. Silibinin slightly changed luciferase activity from reporters containing the miR-21 recognition elements from PTEN-3′UTR and Bcl2-3′UTR in both cell lines. Together these data demonstrated negligible cancer-progression impact of miR-21 and limited roles of miR-21 downregulation in examined silibinin effects, and strengthened the anti-cancer pathways of silibinin other than miR-21downregulation in MCF-7 and T47D cells.     

miR-98-5p靶向PYGO2基因对宫颈癌细胞增殖、迁移和侵袭的影响及其作用机制研究

目的探讨miR-98-5p对宫颈癌细胞增殖、迁移和侵袭的影响以及其作用机制。 方法选取2016年1月至2019年1月郴州市第一人民医院收治的宫颈癌患者的癌组织和癌旁组织;予以miR-98-5p、si-PYGO2及anti-miR-98-5p单独或共培养Siha细胞,记为：miR-NC组、miR-98-5p组、si-NC组、si-PYGO2组、anti-miR-NC组、anti-miR-98-5p组、miR-98-5p+pcDNA组、miR-98-5p+pcDNA-PYGO2组。运用qRT-PCR检测宫颈癌组织和细胞中miR-98-5p和PYGO2 mRNA的表达水平;Western blot检测蛋白表达;MTT法检测细胞增殖活性;Transwell检测细胞迁移和侵袭;将WT-PYGO2、MUT-PYGO2分别与miR-NC、miR-98-5p共转染至Siha细胞中,双荧光素酶报告基因检测实验检测荧光活性。采用方差分析和t检验进行统计学分析。 结果与癌旁组织相比,宫颈癌组织中miR-98-5p表达水平降低（0.98±0.08比0.47±0.05）,PYGO2 mRNA （1.00±0.07比2.43±0.24）和蛋白表达水平（0.27±0.03比0.62±0.05）均升高（P均< 0.001）。与正常宫颈细胞Ect1/E6E7相比,宫颈癌细胞Siha、Hela、Caski中PYGO2 mRNA （0.98±0.09比2.76±0.23、2.46±0.24、2.55±0.21）和蛋白表达水平（0.21±0.03比0.62±0.06、0.51±0.05、0.57±0.06）升高;miR-98-5p的表达水平降低（1.00±0.08比0.34±0.04、0.56±0.05、0.46±0.04） （P均< 0.05）。与miR-NC组相比,miR-98-5p组宫颈癌Siha细胞活性（48 h：0.61±0.05比0.42±0.04,72 h：1.02±0.09比0.59±0.06）、迁移数量[ （112.46±10.27）个比（48.35±4.96）个]及侵袭数量[ （92.47±9.56）个比（39.46±3.52）个]均降低（P均< 0.05）。与si-NC组相比,si-PYGO2组宫颈癌Siha细胞活性（48 h：0.64±0.06比0.46±0.05,72 h：1.05±0.08比0.67±0.06）、Siha迁移数量[ （106.48±9.75）个比（42.16±4.25）个]和侵袭数量[ （87.63±8.11）个比（35.42±6.20）个]均降低（P均< 0.05）;Cyclin D1、MMP-2、MMP-9、MMP-14表达水平降低,p21、p27表达水平升高,差异有统计学意义（P均< 0.05）。与miR-NC组比较,miR-98-5p组转染WT-PYGO2的Siha细胞荧光素酶活性（0.38±0.04比0.99±0.08）降低（P < 0.05）,转染MUT-PYGO2的Siha细胞荧光素酶活性（1.03±0.08比1.01±0.09）差异无统计学意义（P > 0.05）。PYGO2过表达逆转了miR-98-5p过表达对宫颈癌Siha细胞的增殖、迁移和侵袭的抑制作用。 结论miR-98-5p可抑制宫颈癌细胞增殖、迁移和侵袭,其机制可能与其靶向调控PYGO2的表达有关,将可为宫颈癌的预防和治疗提供新靶点。