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.

由于生態(tài)和區(qū)位的不同，北京城市副中心相對應(yīng)的每個鎮(zhèn)變化的建設(shè)用地均不相同，其變化的建設(shè)用地面積大小代表了2030年期限內(nèi)每個鎮(zhèn)的發(fā)展?jié)摿?。因此，城市增長模擬的各鎮(zhèn)發(fā)展?jié)摿A(chǔ)可作為考慮各鎮(zhèn)區(qū)的主觀發(fā)展需求的依據(jù)，在此基礎(chǔ)上可反推出未來通州各鎮(zhèn)集體建設(shè)用地的減量面積，作為未來通州各鎮(zhèn)集體建設(shè)用地指標分配的參考條件。規(guī)劃中，將北京城市副中心各鎮(zhèn)的建設(shè)用地變化量進行計算，得出各鎮(zhèn)的現(xiàn)狀建設(shè)用地變化比例。集體建設(shè)用地指標轉(zhuǎn)換流程如圖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).

患者均展開腹彩超多普勒超聲的檢查，同時進行診斷結(jié)果、手術(shù)病理證實的比較，選擇GE型LOGIQP5與多普勒超聲診斷儀進行，對探頭頻率進行選擇時，需要按照2～9MHz標準進行[2]?；颊咭云脚P位、側(cè)臥位狀態(tài)，保證膀胱充盈。選擇彩色多普勒超聲探頭置于患者腹壁，在對患者胎兒和羊水進行了解后，對胎盤邊緣、子宮頸間關(guān)系進行了解。在對探頭方向進行相應(yīng)調(diào)整后，引導(dǎo)患者正確進行體位變化，仔細觀察胎盤邊緣和子宮頸內(nèi)關(guān)系，以便于進行有效診斷與分類。最后進行胎盤間隙與胎盤實質(zhì)、周邊血流的觀察，同時掌握胎盤植入情況[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).

選擇葡萄酒的人有千萬種因由，但這一路馳騁，從未動搖或懈怠的人真的是鳳毛麟角，連我，也曾一度因為看不清下一個目標而出走過，才意識到我們根本沒有完整地看清這個行業(yè)的路徑，目標也定得太過短小，但他的路徑卻一早就查好了。“路徑真的是想好了，堅持也是一部分吧?！?/p>

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.

作為世界上活動的主體，人類活動對生態(tài)環(huán)境會帶來很大的影響，反過來，自然界的環(huán)境受到破壞會影響人類的正常生活。因此，在建設(shè)生態(tài)林業(yè)時，技術(shù)人員要樹立高度的思想認識，意識到林業(yè)技術(shù)推廣對于生態(tài)林業(yè)建設(shè)來說產(chǎn)生了不容小覷的積極意義。只有在思想上形成積極的態(tài)度，才能更好地與行動結(jié)合，從而落實好生態(tài)林業(yè)建設(shè)的每一個環(huán)節(jié)，在整體水平上提高建設(shè)水平。因此，要做好對技術(shù)人員的思想教育和培訓(xùn)，通過宣傳片的方式來呼吁大家要在思想上對林業(yè)技術(shù)給予足夠的重視，只有這樣才能真正地付諸于實際操作，使林業(yè)技術(shù)水平達到很高的水準，才能夠在生態(tài)林業(yè)建設(shè)中更完美地應(yīng)用林業(yè)技術(shù)。

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.

黃石朝楊露露滿臉堆笑道：“讓你見笑了，小女不懂事，還請包涵。人死了，什么都是空的；高木的病要緊，我看這樣吧，擇日不如撞日，明天我就去把梨花的骨灰遷回來?！睏盥堵兑婞S石這么通情達理，雙腿一軟，要朝他跪了；黃石連忙扶住她道：“高木是我的女婿，應(yīng)該的?！?/p>

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)為相干斑矢量經(jīng)MPWF后輸出.可以看出，極化散射矢量s的MPWF輸出可以分解為上述兩部分的乘積.式(7)中τ1服從歸一化Fisher分布，其PDF為[17]：

機械通氣患者的功能鍛煉無論是對預(yù)后的影響還是對患者戰(zhàn)勝疾病的信心都起到十分重要的作用。長期臥床制動的機械通氣患者會出現(xiàn)嚴重的并發(fā)癥，在重癥監(jiān)護室（ICU）住院超過2周以上，1/3的患者會出現(xiàn)關(guān)節(jié)攣縮[1]。Powers等[2]在一項動物研究中發(fā)現(xiàn)給予機械控制通氣12～24小時后動物膈肌纖維面積開始下降，并且隨著機械通氣時間的延長下降越明顯[3]。延遲脫機拔管是ICU住院時間延長、病死率增加的危險因素[4]。盡早的脫機、早期功能鍛煉對改善患者的預(yù)后起著至關(guān)重要的作用?，F(xiàn)將1例呼吸機依賴患者成功撤機的體會與大家分享。

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，康芳便開始給靜秋張羅對象了。靜秋問康芳：“楚墨哪里不好？”康芳說：“哪里都不好?！膘o秋把康芳的話轉(zhuǎn)給楚墨聽，楚墨聳聳肩，說：“我不就抽了幾根煙、喝了幾瓶酒嗎？”楚墨近乎弱智的樂觀和自信讓他失去了挽救這段感情的最佳時機。

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.

None;None;None;None;None;None.