| Background&objective: Gastric cancer (GC) is one of the mostcommon malignant tumors all over the world. In recent years, although theincidence and mortality of gastric cancer is declining annually, it remainsthe fourth most common malignancy and the second cause of mortalityfollowing lung carcinoma. In China, GC remains the most commonepithelial malignancy and the leading cause of cancer-related mortality.Clinically, although the tremendous advances in the treatments, surgicalresection remains the curative option for GC patients. Similar to other solidtumors, gastric cancer is characterized by local invasion, high regionallymph node metastasis and distant metastasis, which are serious clinicalproblems that lead to recurrence and poor prognosis.AMFR, autocrine motility factor receptor, is a cell surface cytokinereceptor, which involved in numerous physiological and pathologicalprocesses, including cell motility, signal transduction and proteinubiquitination. Recent researches show that AMFR overexpression isclosely linked to aggressive tumor biology and poor outcome formalignancies of the lung, tongue, esophagus, colon, rectum, liver, breast, and skin. Besides inducing cell migration, AMFR also has a dual role as anE3ubiquitin ligase implicated in endoplasmic reticulum-associateddegradation (ERAD).Epithelial-mesenchymal transition (EMT) is a common biologicalprocess, which plays an important role in embryogenesis, chronicinflammation, tissue remodeling, fibrosis and cancer metastasis. It endowsepithelial cells with mesenchymal phenotype, acquiring the abilities toinvade, to disseminate, and to resist apoptosis. E-cadherin, as the mosttypical epithelial marker in EMT and one of the most classical adhesionmolecules, forms a complex with β-catenin to regulate cell adhesion. Andβ-catenin, which is regulated by phosphorylation and ubiquitination-mediated proteasome, is the core factor of Wnt signaling pathway. If Wntsignaling is activated abnormally, β-catenin travels to the nucleus toactivate downstream target genes, leading to tumorigenesis and metastasis.Furthermore, as a sequence-specific and highly effective gene silencingtechnique, RNA inference is widely used in regulation of gene expression,analyses of gene function and signal transduction in recent years.In this research, we investigated the expression of AMFR in a series ofGC tissues to analyse its correlations with clinicopathologic parameters andprognosis, and role in EMT. On this basis, we designed and constructed thelentiviral vector with AMFR shRNA, which specifically silenced theexpression of AMFR in gastric cancer cell line MKN45. Then we detected the changes of malignant biological behaviors and related factors in gastriccancer cell, to explore the possible mechanism. At last we constructed nudemice modal transplated with gastric cancer cell to confirm the above results.We expected that these results would help to elucidate the mechanism ofcarcinogenesis and metastasis in gastric cancer, providing a novelunderstanding and treatment for gastric cancer.Methods:(1) The proteins of AMFR, E-cadherin and N-cadherin wereexamined by immunohistochemistry and Western blot respectively in aseries of GC tissues to analyse its correlations with clinicopathologicparameters and role in EMT. Combined with postoperative follow-upinformation, Kaplan-Meier and Cox regression analyses were applied forthe relationship between AMFR and prognosis.(2) According to AMFR mRNA, four shRNA segments (including anegative control) were specifically designed. Then, these segments weresynthesized and inserted into the shuttle vector pLVT867, which wereidentified by PCR for positive clones and sequencing. Recombinantlentiviral vector with AMFR shRNA were packaged by three-plasmidsystem. After detection of the titers by gradient dilution counting, weinfected gastric cancer cell MKN45with these recombinant lentiviralvectors. The efficiency of infection and optimal MOI were determined byobservation under fluorescence microscope. The expressions of AMFRmRNA and protein were detected by qPCR and Western blot respectively for determination of the optimal RNAi vector.（3）Gastric cancer cell MKN45was infected with recombinantlentiviral vector pLVT-RNAi-AMFR to down-regulate the expression ofAMFR and to establish the cell line silenced AMFR expression stably.After silencing AMFR expression, phenotype of MKN45was observedunder microscope; cell proliferation was examined by MTT assay; cellcycle and apotosis were examined by flow cytometry; migration andinvasion were examined by wound healing assay and millicell chamberassay, respectively; homogeneous and heterogeneous adhesion wereexamined by adhesion assay; colony formation was examined by platecolony assay in vitro; tumorigenicity was examined in nude mice tumormodel in vivo, and pathological changes were evaluated by H-E staining.Expressions of proteins correlated with proliferation, apoptosis, invasionand metastasis were deteced by Western blot.Results:(1) Compared with the normal mucosae, AMFR expressionwas significantly increased in gastric cancer tissues (P<0.05). By contrast,the expression of E-cadherin was significantly decreased in gastric cancertissues; oppositely, the expression of N-cadherin was significantlyincreased in gastric cancer tissues. Correlation analyses showed thatexpression of AMFR negatively correlated with E-cadherin (rs=-0.311),while positively correlated with N-cadherin (rs=0.381). Western blot resultswere consistent with those in IHC assay. Analyses of clinicopathological data showed that overexpression of AMFR was significantly associatedwith invasion depth and lymph node metastasis of GC. Kaplan-Meieranalysis showed that the expression of AMFR significantly correlated witha notable reduction of overall survival and an increased risk of recurrence(P<0.01). Cox regression analysis showed that parallel to advanced TNMstage and distant metastasis, AMFR was an independent predictor for pooroverall survival and recurrence-free survival.(2) Correctness of exogenous sequence, which was inserted intorecombinant lentiviral vector, was confirmed by restriction enzymedigestion and sequencing. The titer of recombinant lentivirus was5.0×108TU/ml. Due to express GFP, gastric cancer cell MKN45infected withrecombinant lentiviral vector was observed with green fluorescence. Theoptimal MOI was40, and the efficiency of infection was over90%at thisMOI. Western blot and qPCR results showed that all recombinant vectorscould down-regulated AMFR expression; and pLVT-dsRNA-1was the bestone among them, whose inhibition ratio was more than75%. So weidentified this vector as the optimal one, and named it pLVT-RNAi-AMFR.(3) Expression of AMFR mRNA and protein were significantlydecreased in gastric cancer cell MKN45infected stably with recombinantlentiviral vector pLVT-RNAi-AMFR; and E-cadherin expression wasdecreased, N-cadherin and Vimentin expressions were decreased,accompanied with epithelial phenotype was restored at a certain extent. After silencing AMFR expression, growth and proliferation of MKN45were inhibited; cell number in G0/G1phase was increased, which wasdecreased in S phase and G2/M phase, accompanied with Cyclin D1expression was decreased (P<0.05). However, apoptosis rate of MKN45was not significantly increased, and Bax and Bcl-2expressions were notsignificantly changed too (P>0.05). Migration rate and invasive cellnumber were also decreased. Adhesion assay showed that homogeneousadhesion was enhanced; whereas, heterogeneous adhesion was weakened;MMP2and MMP9expressions were also decreased correspondingly(P<0.05). Plate colony assay showed that colony formation number wasdecreased in vitro. Nude mice tumor model showed that the growth rate ofxenograft derived from gastric cancer cell MKN45silenced AMFRexpression was slown down, accompanied with weight and volume ofxenografts were less than those in control (P<0.05). H-E staining showedmore relatively loose areas, liquefied necrosis zone, smaller in cell size andnucleus pycnosis; nourish capillary number was also less than that incontrol. Western blot analysis showed that total expression of β-catenin wasdecreased, but no significance (P>0.05). By contast, β-catenin expressionin cyteblast was significantly decreased (P<0.05).Conclusions:(1) The aberrant expression of AMFR was associatedwith certain clinicopathological parameters, invasion depth and lymph nodemetastasis, correlated with poor prognosis in GCs. The enhanced expression of AMFR regulated tumor aggressive progression by involvingin the regulation in EMT process.(2) Recombinant lentiviral vector combined with RNAi technologycould significantly silence the expression of target gene.(3) Silencing AMFR expression could restore epithelial phenotype ofMKN45, enhance homogeneous adhesion, and weaken heterogeneousadhesion; growth and proliferation were arrested at G0/G1phase. Migrationand invasion, colony formation in vitro and tumorigenicity in vivo werealso decreased. Its mechanism would be involved in restoration ofepithelial phenotype and inhibition of Wnt/β-catenin signaling pathway,which could repress the expression of downstream target genes (Cyclin D1and MMPs, etc). |
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