肿瘤相关巨噬细胞相关性miR-99a对子宫内膜癌细胞生长和侵袭的调控作用

背景与目的：肿瘤相关巨噬细胞（tumor-associated macrophage，TAM）浸润与肿瘤进展密切相关，但作用机制尚不明确，因此，探索miR-99a对单核巨噬细胞极化的影响及其对子宫内膜癌（endometrial cancer，EC）细胞生长、侵袭的影响。方法：检测EC组织中巨噬唾液酸蛋白（macrosialin）CD68表达并分析其与临床病理学特征之间的关系；运用人EC细胞系HEC-1B、RL95-2培养上清液诱导人单核细胞U937向TAM（M2型巨噬细胞）分化；将人工合成的miR-99a模拟物片段转染至诱导后的巨噬细胞，转染后运用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）及流式细胞术检测巨噬细胞相关因子CD68、CD163以及巨噬细胞甘露糖受体（mcrophage mannose receptor）CD206表达量变化，并运用酶联免疫吸附法（enzyme-linked immunosorbent assay，ELISA）检测巨噬细胞分泌相关细胞因子IL-12、IL-4和IL-10分泌量变化；将转染miR-99a的诱导后巨噬细胞与EC细胞共培养，运用细胞计数试剂盒（cell counting kit-8，CCK-8）和transwell法检测其对EC细胞增殖和侵袭能力的影响，并初步分析其可能的作用机制。结果：EC组织CD68高表达并与肿瘤肌层浸润及血管生成呈正相关；肿瘤细胞培养上清液成功诱导单核细胞向M2型TAM极化。转染miR-99a后单核细胞组CD68及CD163表达较对照组下降（P＜0.01），而CD206表达差异无统计学意义（P＞0.05），流式细胞术进一步证实上述表达变化；ELISA结果发现，转染miR-99a诱导后巨噬细胞中IL-12分泌增多（P＜0.01），而IL-4、IL-10分泌减少（P＜0.01），提示巨噬细胞向M2型极化受抑制。将诱导后巨噬细胞与EC细胞共培养，共培养后EC细胞增殖侵袭能力较对照组增加，而转染miR-99a模拟片段至诱导后巨噬细胞能够抑制其对增殖（P＜0.01）及侵袭能力的促进作用（P＜0.05）。诱导后巨噬细胞中过表达miR-99a后细胞中mTOR及其通路受到抑制。结论：EC间质巨噬细胞浸润与肿瘤肌层浸润及血管新生相关，miR-99a能够逆转单核细胞向M2表型极化，并抑制EC细胞介导TAM的促EC细胞生长和侵袭作用，其作用可能通过调控mTOR通路产生。     

miR-155 increases stemness and decitabine resistance in triple-negative breast cancer cells by inhibiting TSPAN5

Effective therapeutic targets for triple-negative breast cancer (TNBC), a special type of breast cancer (BC) with rapid metastasis and poor prognosis, are lacking, especially for patients with chemotherapy resistance. Decitabine (DCA) is a Food and Drug Administration-approved DNA methyltransferase inhibitor that has been proven effective for the treatment of tumors. However, its antitumor effect in cancer cells is limited by multidrug resistance. Cancer stem cells (CSCs), which are thought to act as seeds during tumor formation, regulate tumorigenesis, metastasis, and drug resistance through complex signaling. Our previous study found that miR-155 is upregulated in BC, but whether and how miR-155 regulates DCA resistance is unclear. In this study, we demonstrated that miR-155 was upregulated in CD24⁻CD44⁺ BC stem cells (BCSCs). In addition, the overexpression of miR-155 increased the number of CD24⁻CD44⁺ CSCs, DCA resistance and tumor clone formation in MDA-231 and BT-549 BC cells, and knockdown of miR-155 inhibited DCA resistance and stemness in BCSCs in vitro. Moreover, miR-155 induced stemness and DCA resistance by inhibiting the direct target gene tetraspanin-5 (TSPAN5). We further confirmed that overexpression of TSPAN5 abrogated the effect of miR-155 in promoting stemness and DCA resistance in BC cells. Our data show that miR-155 increases stemness and DCA resistance in BC cells by targeting TSPAN5. These data provide a therapeutic strategy and mechanistic basis for future possible clinical applications targeting the miR-155/TSPAN5 signaling axis in the treatment of TNBC.     

miR-548d抑制骨肉瘤细胞的增殖和迁移

目的:探讨miR-548d对骨肉瘤细胞增殖和迁移的影响。方法:通过Real time PCR及Western blot检测miR-548d和KRAS在30例骨肉瘤组织以及293、MG63和U2OS细胞中的表达情况。对30例骨肉瘤组织中miR-548d和KRAS含量的相关性进行分析，随后通过报告基因实验印证miR-548d对KRAS的靶向作用。MG63细胞中分别过表达或沉默miR-548d后，通过Western blot实验分析KRAS的变化情况，MTT实验观察miR-548d对细胞增殖的影响，Transwell实验观察miR-548d对其迁移能力的影响。结果:骨肉瘤组织及细胞中miR-548d表达较低，KRAS表达较高。在骨肉瘤组织中miR-548d与KRAS的表达呈负相关。报告基因实验证明miR-548d可以直接打靶KRAS。Western blot指出miR-548d可以抑制KRAS的表达。最后MTT和Transwell实验指出miR-548d可以抑制MG63细胞的增殖与迁移。结论:miR-548d可以通过打靶KRAS抑制骨肉瘤细胞的增殖和迁移。     

单羧酸转运蛋白1促进胰腺导管癌细胞浸润转移的机制研究

背景与目的：单羧酸转运蛋白1（monocarboxylate transporter 1，MCT1）是细胞转运乳酸、丙酮酸等代谢产物及能量物质的一种重要蛋白质，其在胰腺导管癌中的作用及机制鲜有研究报道。该研究旨在探讨MCT1在胰腺导管癌中的表达及临床病理学意义。方法：纳入78例胰腺导管癌患者的癌组织及癌旁正常组织，运用免疫组织化学技术检测MCT1在癌组织和癌旁正常组织中的表达水平并分析其临床病理学意义。在体外细胞系水平上，我们运用胰腺癌细胞系PANC-1和Capan-1，运用细胞克隆形成实验、细胞划痕和Transwell实验分析沉默MCT1后胰腺癌细胞增殖、迁移和浸润的改变。为明确MCT1的相关作用机制，我们通过生物信息学分析，预测miR-124-3p是MCT1的潜在调控微小RNA；为了进一步验证，我们运用双荧光素酶报告实验分析miR-124-3p对MCT1的调控效果；运用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）分别检测51对新鲜胰腺癌组织中MCT1和miR-124-3p的基因表达并分析两者的相关性。结果：MCT1的阳性表达主要位于细胞膜和细胞质。相比癌旁正常组织，MCT1在胰腺导管癌组织中显著高表达，其表达水平与胰腺导管癌的分化程度、临床分期、淋巴结转移和不良预后具有显著相关性。在体外细胞系水平上，沉默MCT1能够显著抑制胰腺癌细胞系PANC-1和Capan-1的增殖、迁移和浸润；miR-124-3p在胰腺癌组织中显著低表达，并且与MCT1 mRNA的表达具有显著负相关性，能够负调控MCT1的蛋白表达。结论：MCT1是胰腺导管腺癌的致癌基因，miR-124-3p能够负调控MCT1的表达。     

miR-663a inhibits hepatocellular carcinoma cell proliferation and invasion by targeting HMGA2

Hepatocellular carcinoma (HCC) is a highly aggressive solid malignancy throughout the world. Dysregulation of miRNAs play essential roles in HCC progression via aberrant regulation of cell proliferation, apoptosis, as well as metastasis. miR-663a is a poorly investigated miRNA. Whether miR-663a regulates HCC development remains unknown. The aim of the study was to explore the role of miR-663a in HCC development. To determine the expression level of miR-663a in HCC, we analyzed the data from GSE21362 and TCGA. The results showed that miR-663a was significantly down-regulated in HCC tissue compared with adjacent non-tumor tissue. Gain of function and loss of function assays revealed that miR-663a distinctly inhibited cell proliferation, migration and invasion. Mechanistic investigations demonstrated that miR-663a modulated cell functions through targeting and suppressing high mobility group A2 (HMGA2). In addition, overexpression of HMGA2 remarkably attenuated the tumor repressive effect of miR-663a. Taken together, miR-663a inhibits HCC cell proliferation and motility by targeting HMGA2.     

microRNA-622 acts as a tumor suppressor in hepatocellular carcinoma

microRNAs (miRNAs) are important regulators of tumor development and progression. In this study, we aimed to explore the expression and role of miR-622 in hepatocellular carcinoma (HCC). We found that miR-622 was significantly downregulated in human HCC specimens compared to adjacent noncancerous liver tissues. miR-622 downregulation was significantly associated with aggressive parameters and poor prognosis in HCC. Enforced expression of miR-622 significantly decreased the proliferation and colony formation and induced apoptosis of HCC cells. In vivo studies demonstrated that miR-622 overexpression retarded the growth of HCC xenograft tumors. Bioinformatic analysis and luciferase reporter assays revealed that miR-622 directly targeted the 3′-untranslated region (UTR) of mitogen-activated protein 4 kinase 4 (MAP4K4) mRNA. Ectopic expression of miR-622 led to a significant reduction of MAP4K4 expression in HCC cells and xenograft tumors. Overexpression of MAP4K4 partially restored cell proliferation and colony formation and reversed the induction of apoptosis in miR-622-overexpressing HCC cells. Inhibition of JNK and NF-κB signaling phenocopied the anticancer effects of miR-622 on HCC cells. Taken together, miR-622 acts as a tumor suppressor in HCC and restoration of miR-622 may provide therapeutic benefits in the treatment of HCC.     

CircKIF4A靶向调控miR-384表达对卵巢癌SKOV3细胞增殖和凋亡的影响

Objective?To explore the effect of circKIF4A on the proliferation and apoptosis of ovarian cancer SKOV3 cells and its regulatory mechanism on miR-384. Methods?The qRT-PCR method was used to detect the expression of circKIF4A and miR-384 in ovarian cancer tissues and adjacent tissues. si-NC, si-circKIF4A, miR-NC, miR-384 mimics, si-circKIF4A and anti-miR-NC, si-circKIF4A and anti-miR-384 were transfected into SKOV3 cells respectively. The qRT-PCR method was used to detect the expression of circKIF4A and miR-384 in SKOV3 cells. MTT experiment and flow cytometry experiment were used to detect cell proliferation and apoptosis respectively. The dual luciferase reporter experiment was used to detect the targeting relationship between circKIF4A and miR-384. Results?Compared with adjacent tissues, the expression level of circKIF4A in ovarian cancer tissues was increased [(1.00±0.06), (4.28±0.32)] (t=62.915, P＜0.05), and the expression level of miR-384 was decreased [(1.00± 0.05), (0.43±0.03)] (t=61.047, P<0.05). After transfection with si-circKIF4A, the OD value of SKOV3 cells was decreased [(0.75±0.05), (0.41±0.03)] (t=17.493, P＜0.05), and the apoptosis rate was increased [(6.36±0.53)%, (23.19± 2.21)%] (t=22.216, P<0.05). After transfection of miR-384 mimics, the OD value of SKOV3 cells was decreased [(0.73±0.05), (0.47±0.04)] (t=12.182, P＜0.05), and the apoptosis rate was increased [(7.53±0.41)%, (19.11)±1.06)%] (t=30.567, P<0.05). The dual luciferase report experiment confirmed that circKIF4A could adsorb miR-384 and can act as a sponge molecule for miR-384. After co-transfection with si-circKIF4A and anti-miR-384, the OD value of SKOV3 cells was increased [(0.40±0.04), (0.65±0.03)] (t=15.000, P＜0.05), and the apoptosis rate was decreased [(25.20± 2.21)%, (10.37±0.86)%] (t=18.761, P＜0.05). Conclusion?Inhibition of circKIF4A expression could negatively regulate the expression of miR-384, thereby inhibiting the proliferation of ovarian cancer cells and inducing apoptosis.