Effect of miR-184 and miR-205 on the tumorigenesis of conjunctival mucosa associated lymphoid tissue lymphoma through regulating RasL10B and TNFAlP8

INTRODUCTION

Five samples of tumor tissue from patients (4 males and 1 female, mean age 60.6±12.2y, range 40 to 71y, the onset time of 3mo to 6y) with conjunctival MALT lymphoma in this study were collected in Shanghai Changzheng Hospital from July to October in 2016, during anterior open acquisition orbital tumor surgery for MALT lymphoma. The matched adjacent normal conjunctival tissues as controls were obtained and were snap-frozen for further analysis. All patients had no other diseases except MALT lymphoma.

RPMI8226 cells were planted 1.0×10cells per well in 6‐well plates. When culturing to 50% confluence,miR-184 and miR-205 mimics or their negative control were transfected into cells carried by Lipofectamine 2000,respectively. Cell apoptosis was examined by the Annexin V-FITC/PI apoptosis detection kit, and detected by a flow cytometer and Cell Quest Pro version 6.0 software (FACScan;BD Biosciences, Franklin Lakes, NJ, USA).

MATERIALS AND METHODS

miR-184 is involved in regulating the tumorigenesis and metastasis of tumor, as a new member of miRNA family.Previous research showed that the expression level of miR-184 was decreased in various types of tumor. In glioma tumor tissues, the expression of miR‐184 was significantly decreased compared with normal brain tissue. In addition, Liangfound that the level of miR-184 was reduced in central nervous system lymphoma. Meanwhile, the expression of miR-184 was found increased in other types of tumor. miR-184 was extensively expressed in PANC-1 cells, as a pancreatic ductal adenocarcinoma (PDAC) cell line.

由于生态和区位的不同，北京城市副中心相对应的每个镇变化的建设用地均不相同，其变化的建设用地面积大小代表了2030年期限内每个镇的发展潜力。因此，城市增长模拟的各镇发展潜力基础可作为考虑各镇区的主观发展需求的依据，在此基础上可反推出未来通州各镇集体建设用地的减量面积，作为未来通州各镇集体建设用地指标分配的参考条件。规划中，将北京城市副中心各镇的建设用地变化量进行计算，得出各镇的现状建设用地变化比例。集体建设用地指标转换流程如图4所示。

Ocular adnexal lymphoma (OAL) is the most common tumors in the eyes. Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), as a rare form of non-Hodgkin’s lymphoma, is the majority subtype of OAL. The OAL causes a serious threaten to patient health.Therefore, the development of novel target drug is important for patients with OALs. However, the pathological mechanism of OALs is not clearly. MicroRNAs (miRNAs), as a group of short non-coding RNA molecules with 21-25 nucleotides length, regulate target gene translation or degradation through interacting with complementary sites in the 3’ untranslated region (3’UTR) of target mRNAs. Increasing evidences indicated that miRNAs were involved in the pathogenesis of MALT lymphoma. miR-142 and miR-155 regulate the MALT pathogenesis through repressing the target gene TP53INP1. The decreased expression of miR-34a induces the target genes increase, such as FOXP1, p53, and BCL2, to regulate MALT lymphoma development and apoptosis. miR-200 is up-regulated and suppressed the target protein cyclin E2 in conjunctival MALT lymphoma.

RPMI8226 cells were transfected with miR-205 mimics, miR-205 inhibitor or negative control miRNA using INTERFER in transfection reagent following the manufacturer’s instructions (Polyplus). Whole cell extracts for immunoblotting analysis were prepared as previously reported.These antibodies were used for immunoblotting studies:rabbit anti RASL10B (1:500, Abbkine), rabbit anti TNFAIP8(1:1000, Sigma), rabbit anti GAPDH (1:10 000, Sigma), goat anti rabbit IgG-HRP (1:6000, Millipore).

Previous studies indicated that miR-205 and miR-184 were involved in tumor proliferation, apoptosis, invasion, and metastasis. miR-205 acts as tumor activator or suppressor in various types of tumortargeting various genes. The increased miR-205 expression promotes the proliferation,invasion, and migration of nasopharyngeal carcinoma cells.In renal cell carcinoma cells, miR-205 expression is decreased.In renal cell carcinoma cells, PTEN expression is upregulated and p-AKT expression is downregulated after the miR-205 mimics transfection. Previous studies indicated that miR-184 inhibits tumor cells proliferation and invasion in glioma and non-small cell lung cancer cells. Our previous study indicated that the expressions of miR-205 and miR-184 were decreased greatly in MALT tissue compared with control tissue. However, the mechanism of miR-205 and miR-184 regulating MALT lymphoma was not clear.

Transfected cells were gathered and then suspended. For the next step, 5×10or 1×10of the cells were planted onto the upper part of Transwell chambers (Corning, NY, USA), along with or without Matrigel covering (BD Biosciences, SanDiego, CA, USA). Ten percent of FBS was added into the media and placed into the underlying part as a chemo attractant. Twelve or twenty-four hours later, cells migrated or invaded into the underlying part were recorded by an inverted microscope (Olympus, Tokyo, Japan).

患者均展开腹彩超多普勒超声的检查，同时进行诊断结果、手术病理证实的比较，选择GE型LOGIQP5与多普勒超声诊断仪进行，对探头频率进行选择时，需要按照2～9MHz标准进行[2]。患者以平卧位、侧卧位状态，保证膀胱充盈。选择彩色多普勒超声探头置于患者腹壁，在对患者胎儿和羊水进行了解后，对胎盘边缘、子宫颈间关系进行了解。在对探头方向进行相应调整后，引导患者正确进行体位变化，仔细观察胎盘边缘和子宫颈内关系，以便于进行有效诊断与分类。最后进行胎盘间隙与胎盘实质、周边血流的观察，同时掌握胎盘植入情况[3]。

Total RNA was collected by TRIzol reagent (Invitrogen). M-MLV reverse transcriptase (RT; Promega, Madison, WI, USA) was then used to reverse-transcribe. RT primers for miR-205 or random primers (Promega) for TNFAIP8 were from RiboBio(Guangzhou, China). Bio-Rad CFX96 Touch sequence detection system (Bio-Rad Laboratories Inc, Hercules, CA,USA) was used to conduct the quantitative polymerase chain reaction (qPCR) reactions. Platinum SYBRGreen qPCR SuperMix-UDG reagents (Invitrogen) were added into the reaction. Experiments were done three times. U6 or GAPDH were setting as controls. Primers used for qPCR were as followed: Urp-re-R2: 5’-GTGCAGGGTCCGAGGT-3’,miR182-loop-2: 5’-GTCGTATCCAGTGCAGGGTCC GAGGTATTCGCACTGGATACGACAGAGTGTG-3’,miR182-re-F-2: 5’-CGGCGTTTGGCAATGGTAGAAC-3’,miR205-loop-2: 5’-GTCGTATCCAGTGCAGGGTCCGA GGTATTCGCACTGGATACGACCACAGAC-3’, miR205-re-F-2: 5’-CGGCGTCCTTCATTCCACCG-3’, RasL10B-F:5’-GGGGGTACCATGGTCTCCACC-3’, RasL10B-R:5’-GCGGGATCCGCTCGGCCAG-3’, TNFAIP8-F:5’-TTCCATCAGGTGGATTATAC-3’, TNFAIP8-R:5’-AGGTGGCGCTGAATGATTTG-3’.

Human B cell line RPMI8226 (ATCC,Rockville, MD, USA) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and maintained at 37℃ in a humidified atmosphere containing 5%CO. The HEK cell line is a commonly used model cell which has a relatively high transfection rate, while the RPMI8226 cell line, a commonly used human B cell line, is utilized to verify the effect of miRNAs on B cells.

The psiCHECK-2 luciferase reporter plasmid (Promega) carried the cloning RASL10B WT and Mt 3’UTR , TNFAIP8 WT and Mt 3’UTR. Cells were first planted into six‐well plates. One day later, cells were co‐transfected using Lipofectamine 2000 reagent (Invitrogen),with the following: RASL10B WT or Mt 3’UTR reporter plasmids and miR-184 mimic, TNFAIP8 WT or Mt 3’UTR reporter plasmids and miR-205 mimic, and also the control vector pRL-TK (Promega). For the activities of luciferase,results were measured using the Dual-Luciferase Reporter Assay System (Promega). The primers used in luciferase assay as followed: Rasl10b WT-F: 5’-TCGAGCAGCCTTAACTCG ATGGTCCGTCCCTGCCAGGTGCCGC-3’, Rasl10b WT-R:5’-GGCCGCGGCACCTGGCAGGGACGGACCATCGAGT TAAGGCTGC-3’; Rasl10b Mut-F: 5’-TCGAGCAGCCTTA ACTCGATGGAGGCAGCCTGCCAGGTGCGC-3’, Rasl10b Mut-R: 5’-GGCCGCGCACCTGGCAGGCTGCCTCCATCG AGTTAAGGCTGC-3’; TNFAIP8 WT-F: 5’-TCGAGAAGA TGGAGCACTGCTGATTTATGAAGGAAAAAAGAGC-3’,TNFAIP8 WT-R: 5’-GGCCGCTCTTTTTTCCTTCATAAAT CAGCAGTGCTCCATCTTC-3’; TNFAIP8 Mut-F: 5’-TCG AGAAGATGGAGCACTGCTGATTTTACTTCGAAAAAA GAGC-3’, TNFAIP8 Mut-R: 5’-GGCCGCTCTTTTTTCGA AGTAAAATCAGCAGTGCTCCATCTTC-3’; Rasl10b WTF: 5’-CAGCCUUAACUCGAUGGUCCGUCCCUGCCAG GUGCC-3’, Rasl10b Mut: 5’-CAGCCUUAACUCGAUGG AGGCAGCCUGCCAGGUGC-3’; TNFAIP8-WT: 5’-AAGA UGGAGCACUGCUGAUUUAUGAAGGAAAAAAGA-3’,TNFAIP8-Mut: 5’-AAGAUGGAGCACUGCUGAUUUUAC UUCGAAAAAAGA-3’.

All statistical analysis were completed by the SPSS 15.0 statistical software (SPSS Inc., Chicago,IL, USA). Difference of measurement data was compared with-test or one-way ANOVA.<0.05 were considered statistically significant.

RESULTS

RNA was extracted from tumor tissues and adjacent tissues of 5 patients with MALT, and the mRNA expression of miR-184 and miR-205 was detected by qRT-PCR. The results showed that the expression of miR-184 and miR-205 mRNA were significantly downregulated in the lymphoma tissues compared with the adjacent tissues (<0.001; Figure 1).

Ⅰn order to study the effect of miR-184 and miR-205 on the proliferation of lymphoma cells,miR-184 mimics and corresponding negative controls were transfected into the RPMI8226 cells. The CCK8 kits were used after transfection of 24, 48, 72, and 96h, and the value with 450 nm wavelength was detected. The results showed that the transfection of miR184 mimics notably inhibited the proliferation rate of RPMI8226 cells than the negative group.A similar result was observed when miR-205 mimics was transfected into the RPMI8226 cells. The results showed that the proliferation rate of RPMI8226 cells was significantly inhibited after transfected with miR-205 mimics than with the negative group. These results indicate that miR-184 and miR-205 notably inhibit the proliferation of RPMI8226 cells.

Then, the effect of miR-184 and miR-205 on the apoptosis of lymphoma cells was explored by flow cytometry. The results showed that the apoptosis rate was 0.06%±0.006% in normal RPMI8226 cells group, and the rate was 1.2%±0.73%in negative control group. Apoptosis rate was 12.4%±0.32%in miR-184 mimics treated group, and the rate of the miR-205 mimics treated group was 17.1%±0.42%. Compared with normal group and negative control group, miR-184 and miR-205 mimics induced the apoptosis rate of RMPI8226 cells increased significantly (Figure 2;<0.001).

Previous study has shown that RasL10B may be a downstream target protein of miR-184 which participates in the pathogenesis of MALT lymphoma.We detected the expression level of RasL10B in MALT lymphoma tissues by real-time qPCR and Western blotting.The results revealed that RasL10B mRNA and protein in MALT lymphoma were significantly decreased compared para-cancerous tissue (Figure 5A-5C). The results of luciferase test showed that after transfection of miR184 mimics, the luciferase expression was significantly reduced (<0.01) after transfection of the wild type RasL10B gene 3’UTR compared with the control group and the mutant group, indicating that miR184 has direct regulation on 3’UTR of the RasL10B gene(Figure 5D). In terms of the effect on the control group, the protein expression level of RasL10B was raised in RPMI8226 cells transfected to miR-184 mimics. However, miR184 inhibitor obviously decreased the expression level of RasL10B protein (<0.05; Figure 5E, 5F). These results suggested that miR184 can promote the gene expression of RasL10B in MALT lymphoma and RPMI8226 cells.

Furthermore, we detected the effect of miR-184 and miR-205 on the invasion of RPMI8226 cells. The results showed that comparison to the negative group, the number of invaded cells in the miR-184 inhibitor treated groups was 142.2%±21.3%, while the number in mimics treated group was only 68.1%±12.7% (Figure 4A, 4C). After transfection of 72h, compared to the negative control group, the number of invaded cells in the miR-205 mimics treated group was only 59.2%±12%, while the number of miR205 inhibitor groups through the cells was 143.4%±24.1% in the control group(Figure 4B, 4D). These results suggested that miR-184 and miR-205 attenuated the ability of migration and invasion of RPMI8226 cells.

The effect of miR-184 and miR-205 on migration of RPMI8226 cells were examined by Transwell assay. The results showed that comparison to the negative group (100%), the number of migrated cells was the 118%±4.9% in the miR-184 inhibitor treated group. However,the number of migrated cells only was 61.4%±12.7% in the miR-184 mimics treated group (Figure 3A, 3C). Comparison to the negative control group (100%), the number of migrated cells in miR-205 inhibitor treated group was 110.7%±4.5%,while the number of migrated cells in the miR-205 mimics treated group was only 53%±11.7% (Figure 3B, 3D).

选择葡萄酒的人有千万种因由，但这一路驰骋，从未动摇或懈怠的人真的是凤毛麟角，连我，也曾一度因为看不清下一个目标而出走过，才意识到我们根本没有完整地看清这个行业的路径，目标也定得太过短小，但他的路径却一早就查好了。“路径真的是想好了，坚持也是一部分吧。”

Previous study has shown that miR-205 participates in the tumor pathogenesis through regulating TNFAIP8. The expression level of TNFAIP8 mRNA and protein in MALT lymphoma were remarkably decreased compared with adjacent controls (Figure 6A-6C). The results of luciferase test showed that the luciferase expression significantly decreased after adding wild type TNFAIP8 gene 3’UTR compared with the control group and the mutant group (Figure 6D).The expression of TNFAIP8 protein was downregulated in RPMI8226 cells along with the transfection of miR-205 inhibitor by comparison to the negative group (Figure 6E, 6F).These results suggested that miR-205 can promote the gene expression of TNFAIP8 in MALT lymphoma and RPMI8226 cells.

DISCUSSION

Dysfunctions of miRNAs are associated with many fields of tumor biology, such as tumor proliferation, apoptosis, and metastasis. miRNAs play bi-directional role as oncogenes or anti-oncogenes. Through previous research, we found that the expression level of miR-184 and miR-205 were abnormal in the MALT lymphoma tissue. In present study, we found a significantly decreased expression level of miR-184 and miR-205 in MALT tissues. In gain- and loss-of-function assay, miR-184 and miR-205 mimics suppressed the cellular proliferation, migration, and invasion of RPMI8226 cells and promoted the apoptosis of RPMI8226, while miR-185 and miR-205 inhibitor increased cellular migration and invasion.Luciferase assay and gain- and loss- of function assay indicated that miR-184 directly regulated target gene RasL10B and miR-205 regulated target gene TNFAIP8 in MALT lymphoma tissue and RPMI8226 cells. miR-184 and miR-205 could be a potential tumor suppressor of MALT lymphoma.

作为世界上活动的主体，人类活动对生态环境会带来很大的影响，反过来，自然界的环境受到破坏会影响人类的正常生活。因此，在建设生态林业时，技术人员要树立高度的思想认识，意识到林业技术推广对于生态林业建设来说产生了不容小觑的积极意义。只有在思想上形成积极的态度，才能更好地与行动结合，从而落实好生态林业建设的每一个环节，在整体水平上提高建设水平。因此，要做好对技术人员的思想教育和培训，通过宣传片的方式来呼吁大家要在思想上对林业技术给予足够的重视，只有这样才能真正地付诸于实际操作，使林业技术水平达到很高的水准，才能够在生态林业建设中更完美地应用林业技术。

The abnormal expression of miR-184 and miR-205 in various types of tumor participates in tumor pathogenesis and progression. This study demonstrated that the expression of miR-184 and miR-205 in MALT lymphoma tissue were decreased compared with adjacent tissue.

The Committee of Second Military Medical University biotechnology ethics reviewed. Samples were got followed the Helsinki Declaration. The samples were from conjunctival MALT lymphoma patients who signed the informed consent.

黄石朝杨露露满脸堆笑道：“让你见笑了，小女不懂事，还请包涵。人死了，什么都是空的；高木的病要紧，我看这样吧，择日不如撞日，明天我就去把梨花的骨灰迁回来。”杨露露见黄石这么通情达理，双腿一软，要朝他跪了；黄石连忙扶住她道：“高木是我的女婿，应该的。”

miR-205, as a conserved gene among multifarious species,is associated with tumor pathogenesis and progress. The expression of miR-205 is altered in different types of tumor tissue. In renal cell carcinoma, the expression level of miR-205 was downregulated compared with normal renal cells. Childsfound that the low-level of miR-205 expression was a prognostic factor of head and neck squamous cell carcinoma.In other hand, previous studies showed that the expression level of miR-205 increased in some tumors. In human endometrial endometrioid carcinoma, the level of miR-205 was significantly increased. The altered expression of miR-184 and miR-205 suggest that miR-184 and miR-205 play various roles in different types of cancer.

miR-184 and miR-205 has opposite effect in various tumors through acting as an oncogene or an anti-oncogene. In some types of tumors, miR-184 and miR-205 promote the tumorigenesis and metastasis. It has been recently reported that miR-184 expression was increased in the osteosarcoma cells, and the miR-184 mimics enhanced the proliferation of osteosarcoma cells. Sufound that when transfected with the miR-205 mimics, the proliferation, migration,and invasion ability of endometrial carcinoma tumor cells was upregulated. Nevertheless, our study demonstrated that miR-184 and miR-205 may be as a suppressor in the tumorigenesis and development of MALT lymphoma. In gainand loss-of-function assay, miR-184 and miR-205 mimics reduced proliferation, migration and invasion of RPMI8226 cells, but promoted cellular apoptosis. Meanwhile, inhibition of miR-184 and miR-205 promoted the cellular migration and invasion. Our study is according with early studies in central nervous system lymphoma, renal cell carcinoma, and other tumors. Previous study showed that exogenous miR-184 suppressed the cell survival and invasion of central nervous system lymphoma, but miR-184 inhibitor could reverse the process. Wangfound miR-205 mimics promote renal cell carcinoma cells apoptosis and inhibited the cellular proliferation and metastasis. In addition, the expression of miR-205 suppressed the proliferation, migration and invasion of gastric cancer cells.

其中，x=Tr(P-1Y)为相干斑矢量经MPWF后输出.可以看出，极化散射矢量s的MPWF输出可以分解为上述两部分的乘积.式(7)中τ1服从归一化Fisher分布，其PDF为[17]：

机械通气患者的功能锻炼无论是对预后的影响还是对患者战胜疾病的信心都起到十分重要的作用。长期卧床制动的机械通气患者会出现严重的并发症，在重症监护室（ICU）住院超过2周以上，1/3的患者会出现关节挛缩[1]。Powers等[2]在一项动物研究中发现给予机械控制通气12～24小时后动物膈肌纤维面积开始下降，并且随着机械通气时间的延长下降越明显[3]。延迟脱机拔管是ICU住院时间延长、病死率增加的危险因素[4]。尽早的脱机、早期功能锻炼对改善患者的预后起着至关重要的作用。现将1例呼吸机依赖患者成功撤机的体会与大家分享。

In various types of tumor, miR-184 and miR-205 exerts their function through different target genes. To investigate the mechanism by which miR-184 and miR-205 exert their functions in MALT lymphoma, possible downstream target genes were focused. RasL10B is a member of Ras family,but its function remained unclear. Zoufound that RasL10B was downregulated in breast tumor cells. Our results displayed similar results. In present study, the expression level of RasL10B was reduced in MALT tissue and RPMI8226 cells. Luciferase assay indicated miR-184 could directly regulate RasL10B 3’UTR. Furthermore, after treatment of miR-184 mimics, the RasL184 expression was inhibited in RPMI8226 cells, and miR-184 inhibitor reversed this process.These results suggest RasL10B as miR-184 downstream target protein, had suppressor potential in tumorigenesis and development. Excepted RasL10B, miR-184 modulated tumorigenesisregulation of Wnt/β‐catenin signaling pathway in osteosarcoma.

楚墨回去以后，康芳便开始给静秋张罗对象了。静秋问康芳：“楚墨哪里不好？”康芳说：“哪里都不好。”静秋把康芳的话转给楚墨听，楚墨耸耸肩，说：“我不就抽了几根烟、喝了几瓶酒吗？”楚墨近乎弱智的乐观和自信让他失去了挽救这段感情的最佳时机。

The deletion of TNFAIP8, which is expressed in lymphoid tissues, leads to multiorgan inflammation, splenomegaly, and premature death. Recently studies reported TNFAIP8 played an important role in tumor cell survival and apoptosis.Our results displayed that TNFAIP8 was decreased in MALT tissue and RPMI8226 cells compared with control group.Additionally, after treatment of miR-205 mimics, the TNFAIP8 expression was inhibited in RPMI8226 cells, and miR-205 inhibitor reversed this process. These results suggest that TNFAIP8 is miR-205 downstream target protein.

It has reported that miR-205 inhibited tumorigenesis and metastasisPTEN/AKT pathway. In addition, miR-205 has been reported to regulate Smad4 and ubiquitin specific peptidase 7 in human non-small cell lung cancer and hepatocellular carcinoma. As we have revealed the role of miR-184/RasL10B and miR-205/TNFAIP8 in regulation of RPMI8226 cells, further investigation is needed to study the function of miR-184 and miR-205in the tumor tissue and the downstream pathway regulated by miR-184 and miR-205.In conclusion, we revealed that the level of miR-184 and miR-205 mRNA in MALT was downregulated, and miR-184 and miR-205 analogues promote apoptosis of lymphoma RPMI8226 cells and attenuated the proliferation, migration,and invasion of RPMI8226 cells. The mRNA and protein level of RasL10B and TNFAIP8 were downregulated in MALT lymphoma tissue. miR-184 had direct regulation on the target gene RasL10B. Meanwhile, that the target gene TNFAIP8 was directly regulated by miR-205. Up of these suggested that miR-184 and miR-205 were presented as potential targets for the treatment of MALT lymphoma.

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