Function And Mechanism Study Of Long Non-coding RNA Lnc-LEMGC, Oncogene KIF26B In Gastric Cancer

Gastric cancer, heading the list of digestive system malignant tumors, is common worldwide now. Although in recent years, the diagnosis and treatment of gastric cancer has improved, the prognosis of patients with gastric cancer is still poor, and the 5-year survival rate is below 30%. Invasion and metastasis of gastric cancer is the leading cause of death in patients with gastric cancer. The process of gastric cancer invasion and metastasis is a result of abnormal genes and signaling pathways, which formed a complex network to affect the occurrence of tumor metastasis on different levels. To explore the mechanism of metastasis of gastric cancer and to find new therapeutic targets have become current research focus. Previous studies reported that the molecular basis of gastric cancer occurrence and progression, especially the activation of proto-oncogenes or (and) the inactivation of tumor suppressor genes is closely related to tumor metastasis. With the development of sequencing technology, miRNA and long noncoding RNA, which account for the higher percentage of non-coding RNA, gradually attract the attention of the researchers. MiRNAs has now been confirmed to play an important regulatory role in gastric cancer invasion and metastasis process. However, the known functional lncRNAs are still minority, and esearch on metastasis related lncRNAs in human gastric cancers is a rare event.In this study, we use lncRNA microarray, high throughput screening differential expression genes of nontumorous gastric tissues, primary gastric cancer tissues without metastasis, and primary gastric cancer tissues with metastasis. The lncRNA microarray can detect lncRNA and mRNA expression level at the same time. Through bioinformatics analysis and classical molecular experiments, we studied the functions of long non-coding RNA and encoding genes KIF26B on gastric cancer cell invasion and metastasis in the clinical sample level, cell experiment level and nude mouse models.PART ONE Long noncoding RNA lnc-LEMGC promots proliferaton and inhibits metastasis of gastric cancer cellsBased on the lncRNA expression profile, we screened the differential expression lncRNAs associated with gastric cancer metastasis. The results were further validated in other independent samples by Real-time PCR. The fine genetic information analysis of lnc-LEMGC was carried out using bioinformatics and related experiments, combing with functional experiments, we finally identified clearly showed clearly that the lncRNA was related with gastric cancer metastasis in vivo and in vitro. We used biotin labeling lnc-LEMGC for RNA pull-down with protein components of gastric cancer cell, and the direct combined proteins were identified by affinity purification and mass spectrometry. Results showed that lnc-LEMGC were associated with DNA-PKcs in vivo. The Western blot was further validated this notion. The whole genome microarray showed lnc-LEMGC coordinates with DNA-PKcs to regulate the downstream target genes. The results were as followed:(1) Based on the lncRNA microarray screening technology, we found that the lncRNAs expressed differentially in nonmetastatic gastric cancer tissues, metastatic cancer tissues and nontumor mucosal tissues. Metastatic gastric cancer tissue specific lncRNA expression spectrum can differentiate metastatic gastric cancer and nonmetastatic gastric cancer tissues. Using data analysis and large sample validation, we screened an obviously differentially expressed lncRNA, named lnc-LEMGC, for further function and mechanism research. Real-time PCR revealed that the Inc-LEMGC expression level was higher in nonmetastatic gastric cancer tissues than that in metastatic cancer tissues. These results were consistent with the lncRNA microarray.(2) Integrated search about Inc-LEMGC genetic characteristics in online website showed that Inc-LEMGC was located on chromosome 1 q-arm, with little conservation between species and no encoding proteins ability. RNA in situ hybridization technique showed that Inc-LEMGC mainly expressed in the cytoplasm, with only a small amount of expression in cell nucleus in gastric cancer cells and gastric tissue sections.(3) Transwell Chambers, wound healing, and matrix invasive experiments showed that overexpression of Inc-LEMGC can reduce SGC 7901, BGC 823 gastric cancer cell migration and invasion. On the other hand, inhibition of Inc-LEMGC expression level promoted the gastric cancer cell migration and invasion ability. MTS and EdU experiment revealed that Inc-LEMGC had no effect on proliferation of gastric tumor cells. Experiments in vitro supported that Inc-LEMGC inhibited gastric cancer cell metastasis.Next, we used nude mice models to further verify the cell function conclusion. Two major groups of animals were used, the first group were injected with gastric cancer cells from stable expression of Inc-LEMGC SGC 7901 cell line, while the second group were used with virus-system built Inc-LEMGC knockdown BGC 823 gastric cancer cell line. The animals living fluorescence tracer technology revealed that Inc-LEMGC promoted the growth of subcutaneous transplantation tumor in nude mice, but inhibited gastric cancer cells metastasis in nude mice in vivo. In Inc-LEMGC knockdown BGC 823 cell line group, nude mice subcutaneous tumors were significantly smaller in volume than those in control group, and necrosis was observed in the center of the tumors. This phenomenon was also observed in the SGC 7901 cell line group, we speculate that Inc-LEMGC may affect the tumor blood supply, leading to tumor center necrosis caused by ischemia.(4) The full sequence of Inc-LEMGC was amplified by Inc-LEMGC specific primers and was cloned into pcDNA3.1 (+) vector. And the Inc-LEMGC antisense was constructed into plasmid as the RNA pull-down negative control. Biotin labeling Inc-LEMGC was synthesized by transcription in vitro. After combination with Inc-LEMGC, protein was disposed and analyzed affinity purification and mass spectrometry. From the result of mass spectrometry, combined with the reported literature of related genes, DNA-PKcs was identified as the Inc-LEMGC binding protein. Western blot further verified the protein. Then by knocking down the DNA-PKcs in gastric cells for transwell assay, we found that inhibition the expression of DNA-PKcs can improve gastric cancer cell migration and invasion ability. These results showed that Inc-LEMGC played a role in regulating gastric cancer cell migration and metastasis by binding protein DNA-PKcs.(5) MRNA expression microarray screened differentially expressed genes in the gastric cancer cell line SGC7901, which had been transfected with pPEGF-Inc-LEMGC and empty vector respectively for 48h. Kyoto Encyclopedia of Genes and Genomes (KEGG) website was used for gene pathway analysis. Combining with the existing gene functions, downstream target genes regulated by Inc-LEMGC were preliminarily determined. MRNA expression microarray analysis showed that differentially expressed genes focused on ErbB signaling pathways (especially TGFA-ErbB1-Src-FAK signal pathways), which were associated with cell migration and invasion in cancer. Multiple molecules (including ErbB1, Src, FAK, Ras, MEK, Myc, etc.) obviously changed in the expression. The Real-time PCR and Western blot have verified the regulation function of Inc-LEMGC in the signal pathways.At the same time, we observed that as the expression level of Inc-LEMGC rose, so did that of VEGFA. Real-time PCR and Western blot have verified the regulation function of Inc-LEMGC in the gastric cancer cell lines. The phenomenon was further validated using nude mice subcutaneous tumors. The results suggested that Inc-LEMGC promoted gastric cancer cell proliferation by regulating tumor angiogenesis through VEGFAThe above research results illuminated the lncRNA coordinated with DNA-PKcs to inhibit the metastasis of gastric cancer cells by regulating ERBB signaling pathway. Meanwhile, lncRNA promoted angiogenesis via VEGFA to facilitate gastric cancer cell proliferation. This study not only expanded the lncRNA research in gastric cancer, but also established foundation platform for future gastric cancer metastasis related lncRNA research.PART TWO KIF26B promotes proliferation and metastasis by activating the VEGF signaling pathway in gastric cancerIn the first part, we studied the molecular mechanisms how the Inc-LEMGC regulated the progress of gastric cancer. Firstly, we detected whether Inc-LEMGC cis-regulated the expression of adjacent gene SMYD3 and KIF26B. There was no correlation of Inc-LEMGC and the adjacent protein-coding genes (SMYD3 and KIF26B). However, the expression level of KIF26B in metastatic gastric cancer tissues was significantly higher than the nonmetastatic gastric cancer tissues. Therefore, we further studied the biological functions of protein encoding gene KIF26B in gastric cance. The results were as followed:(1) Quantitative PCR was used to measure K.IF26B expression in frozen human gastric tissues and the results showed that KIF26B transcripts were up-regulated in metastatic gastric carcinoma tissues compared with those in nonmetastatic gastric cancer tissues. Furthermore, expression of KIF26B was analyzed in seven human gastric cancer cell lines (MKN28, MKN823, MKN45, SGC7901、MGC803, KATO III, AGS) and one immortalized human gastric mucosal epithelial cell line (GES-1). KIF26B expression was weakly detected in the immortalized gastric mucosa cell line, but its expression was substantially increased in gastric cancer cell lines. In addition, KIF26B expression was up-regulated in poorly differentiated and metastatic gastric cancer cell lines compared to modestly and well differentiated cell lines. Next, KIF26B protein was confirmed immunohistochemically in an independent panel of paraffin-embedded samples with follow-up data. KIF26B expression was up-regulated in a stepwise fashion from nontumorous gastric tissues, primary gastric cancer tissues without metastasis, via primary gastric cancer tissues with metastasis, to the secondary lymph node metastatic foci. Increased expression of KIF26B was correlated with larger tumor size, positive lymph node metastasis, or distant metastasis and poorer prognosis.(2) MTS and EdU assays suggested that KIF26B knockdown significantly inhibited proliferation of BGC 823 and SGC 7901 cells, whereas KIF26B overexpression dramatically enhanced cell growth. In addition, a clonogenic assay indicated that suppression of KIF26B expression decreased colony formation efficiency of BGC 823 and SGC7901 cells. And apoptosis assay revealed no effect of KIF26B on apoptosis of gastric tumor cells.To test the effect of KIF26B in vivo, xenograft nude mouse model experiments were performed and KIF26B expression in the LV-shRNA-KIF26B group was decreased more than that in the negative control group. Xenograft tumor growth in the LV-shRNA-KIF26B group was significantly slower than that in negative control group. Immunohistochemistry for Ki67 revealed tumor cells in the control group had more proliferative activity than those in the LV-shRNA-KIF26B group.(3) Transwell chamber and matrigel invasion assays indicated that elevated KIF26B could facilitate invasion and metastasis of gastric cancer cell lines SGC7901 and BGC823. On the contrary, decreased KIF26B suppressed those abilities. Migration and invasion assays showed that KIF26B-inhibited cells had less migratory and invasive capacity. Conversely, KIF26B overexpression enhanced migration and invasion activity in BGC823 and SGC7901 cells.In subcutaneous implanted models, tumors in the LV-shRNA-KIF26B group were noninvasive or well-encapsulated. However, tumors in the negative control group displayed local invasion, with islands of cancer cells invading the muscle tissues. RFP imaging indicated that decreased expression of KIF26B suppresses the ability of BGC823 tumor cells to spread to the lung or other organs after tail-vein injection. Only lung metastases were observed in the LV-shRNA-KIF26B group (n=5). However, metastatic foci were found in lung (n=6), kidney (n=1) and lymph nodes (n =1) in the negative control group. The lungs in the LV-shRNA-KIF26B group contained 24.76±3.87 metastatic gastric cancer foci, whereas the control group had 44.31±6.38 metastatic foci as viewed under the microscope. Moreover, the metastatic foci in the control groups had greater volumes than those in the LV-shRNA-KIF26B group.(4) Given the functional role of KIF26B in gastric cancers, the molecular events causing aberrant KIF26B expression were investigated. Based on a prediction algorithm TargetScan (www.targetscan.org) and previous descriptions on the functions of cancer related miRNAs, we screened miR-29a, miR-93, miR-106a, miR-106b, miR-372 as putative miRNAs targeting KIF26B. Luciferase assays, Real-time PCR, Western blot, and double immunofluorescent staining revealed that miR-372 rather than the other four candidates significantly reduced luciferase-KIF26B 3’-UTR reporter activity. These data suggested that KIF26B expression could be negatively regulated by miR-372.(5) To characterize the overall cellular response to KIF26B silencing, gene expression microarray profiling was performed in SGC7901 cells. Compared with negative control transfected cells,1076 genes were up-regulated and 882 were down-regulated. Pathway analysis suggested that several significantly enriched gene categories were involved in pathways related to the VEGF signaling pathway. Quantitative PCR and Western blot confirmed that VEGFA, VEGFR, PXN, FAK, PI3KCA, BCL2, CREB1, and Src mRNA expression were suppressed in the KIF26B siRNA group.VEGFA activation is an initial step for activating the VEGF pathway, so weather KIF26B cooperates with VEGFA to activate this pathway was investigated. VEGFA mRNA and protein expression was obviously lower in xenograft tumor nodules of the KIF26B knockdown group compared with that of the negative control group. Then, using human gastric cancer samples, we identified explicit co-expression relationship between KIF26B and VEGFA in gastric cancer tissues. (r= 0.087, p= 0.007). Taken together, these data consistently suggest KIF26B promotes proliferation and metastasis via the VEGF pathway, and that VEGFA, VEGFR, PXN, FAK, PI3KCA, BCL2, and CREB1 are involved.In conclusion, we identified KIF26B as an important oncogene that activates the VEGF signaling pathway. Targeting KIF26B may be a favorable therapeutic strategy for the future treatment of patients with gastric cancer.