Loss Of TINCR Expression Promotes Proliferation,Metastasis Through Regulating EpCAM Cleavage In Colorectal Cancer

Background and ObjectiveColorectal cancer (CRC) is a common digestive system tumor, in our country, the incidence rate is increasing year by year. In recent years, although the diagnosis and treatment technology and chemotherapy drugs continue to update, great improvement had been achieved on the treatment effect of patients with colorectal cancer, but the prognosis of patients with colorectal cancer has not significantly improved since most CRC had been metastasis when patients found the disease. Therefore, it is important clinical value and social significance to:study the mechanism of colorectal cancer metastasis, search for new molecular markers and therapeutic targets, improve the survival rate of patients through early intervention and individualized treatment.Long noncoding RNA (LncRNA) is a kind of noncoding RNA, which is more than 200nt, and is lack of the open reading frame. LncRNA regulates genes expression mainly through epigenetic, transcriptional and post transcriptional regulation. In colon cancer tissues and cells, lncRNA regulates the proliferation and metastasis in varieties mechanism. Firstly, lncRNAs can modify the stability of chromatin. HOTAIR cooperating with Polycomb complex PRC2 could reprogram chromatin organization and promote cancer metastasis in breast cancer, and colorectal cancer. CCAT2 is highly overexpressed in microsatellite-stable colorectal cancer, and promotes tumor growth, metastasis and chromosomal instability. In addition, several lncRNAs function as transcriptional activators or repressors in CRC. LncRNA-RMST maintain the sternness of the nervous system stem cell by formation a complex with Sox2 and binding to the promoter region of the transcription factor. The fact that overexpression of lncRNA-ROR in wild HCT116 cells could significantly decrease the expression of p53 indicates that ROR is a strong repressor of p53. Furthermore, the ability of lncRNA to interact with signal transduction pathways allows them to regulate the function of cancer cells. Colorectal cancer-associated lncRNA (CCAL) promotes CRC progression by targeting AP-2 a, and then activated the Wnt/β-catenin pathway. LncRNA-PVT-1 decrease the proliferation and invasion capabilities by acting the TGF-β signal pathway and the apoptotic signals in CRC cells. Thus, lncRNA is involved in the regulation of gene expression in multiple levels, including proliferation, differentiation and apoptosis of tumor cells, and lncRNAs are involved in the whole process of tumor development and metastasis.Kretz found that Terminal differentiation-induced lncRNA (TINCR) was up regulated in human high differentiated skin tissues. TINCR could induce cell differentiation by maintaining the stability of differentiated gene mRNA through combining with staul protein. Meanwhile, their also found that when si-TINCR, some other genes mRNA was upregulated instead. This result indicated that TINCR may play its function through other mechanisms. Currently, little is known about the function and the mechanism of TINCR in CRC. Our previous experiments confirmed that LncRNA-TINCR was down regulated in colorectal cancer tissues, and negatively correlated with tumor biological behavior; Functional experiments results show that down regulated TINCR could promote tumor cells invasion and metastasis in CRC. On these basis, in study, we will further explore the role and mechanism of TINCR in the proliferation and metastasis in colorectal cancer, in order to provide a new target for clinical diagnosis, treatment and prognosis evaluation.Methods1. Downregulation of LncRNA-TINCR correlated with CRC progression.Bioinformatics predicted the expression of TINCR in oncomine database. Real time fluorescence quantitative RT-PCR (RT-qPCR) was used to detect the expression of TINCR in 44 colorectal cancer tissues and their corresponding adjacent tissues; The expression of TINCR in colorectal cancer cell lines, such as SW480, HCT116, HT29, LS174T, RKO, SW620 and LoVo were detected by RT-qPCR. The expression of TINCR in HCT116, SW480 cytoplasm and nuclear were detected by RT-qPCR.2. TINCR inhibits CRC cells proliferation, migration and metastasis.(1) Lentiviral constructs repressing TINCR was packaged using the genechem lentiviral expressing system; Pseudovirus particles were subsequently used to infect CRC cells as normal control (NC) groups. HCT116 and SW480 cell lines stably suppressing TINCR were generated. And the cells were labelled with HCT116/shTINCR and SW480/shTINCR.(2) The prime 5.0 software was used to design the primes of TINCR; RT-PCR was used to amplify the whole sequence of TINCR, while the normal colon mucosa was used as template; and the sequence was subcloned into pcDNA3.0 vector, and the vectors were transfected into RKO and LoVo cells to generate stably expressing TICNR cell lines. And the cells were labelled with RKO/oeTINCR and LoVo /oeTINCR.(3) The CCK-8 cell proliferation assays, plate colony formation assays, Xenograft tumors generated by subcutaneous injection were used to assess the effect of TINCR on cell proliferation capacity in vivo and in vitro; Wound healing assay, Transwell migration assay were used to detect cell migration capacity in each group; Transwell invasion assay; Xenograft tumors generated by Tail vein injection were used to assess the effect of TINCR on colorectal cancer cell invasion and metastasis.3. TINCR specifically binds to EpCAM and regulates its proteolysis.(1) RT-qPCR, Western blot were used to detect the EpCAM mRNA and protein level in HCT116 and SW480 cells after knockout the TINCR.(2) Western blot was used to detect the half-time of EpCAM in HCT116 and SW480 cells after knockout the TINCR.(3) RNA pull down, RNA immunoprecipitation were used to detect the combination of TINCR and EpCAM.(4) Psen2 overexpression vector were constructed, and then transfected into HCT116/shTINCR cell and HCT116/oeTINCR cell; Western blot were used to detect the EpCAM and EpICD protein level.(5) Immunohistochemistry and Western blot were used to detect the expression of EpICD, which is the intracellular fragment of EpCAM, in colorectal cancer tissues and corresponds normal tissues. Statistics methods were used to analysis the relationship between the expression of TINCR and EpICE.4. Downregulation of TINCR activates WNT/β-catenin pathway in CRC.(1) TOP/FOP vectors were amplified and extracted, and then transfected into HCT116/shTINCR and SW480/shTINCR cells; luciferase report system was used to detect the effect of TINCR knocked down on the wnt/β-catenin pathway.(2) Proteins and mRNAs in HCT116, SW480 cells which knocked out TINCR expression were collected. Western blot and RT-qPCR were used to detect WNT/β-catenin pathway relative genes protein and mRNA levels respectively.(3) co-IP was used to detect the combination of EpICD and β-catenin in HCT116/shTINCR and SW480/shTINCR cells.5. c-Myc represses the expression of TINCR through inhibiting the transcriptive activity of Spl(1) Through UCSC, ESEMBLE and other websites, bioinformatics methods were used to predict the TINCR promoter sequence and the combination of related transcription factors.(2) Sp1 expression vector was constructed, and then transfected into HCT116 and SW480 cells. RT-QPCR was used to detect the expression of TINCR. Promoters fragment containing spl bind sites were amplified, while also amplified the promoter sequence which sp1 binding site were muted, and then subcloned these sequence to the pGL3-basic vector. Co-transfected sp1 overexpression vector with TINCR promoter luciferase reportor vectors in HEK293A, HCT116, and SW480 cells. Dual luciferase report system was used to detect the effect of sp1 on the TINCR promoter sequence.(3) CHIP was used to detect the combination of Spl with TINCR promoter sequence.(4) Spl expression vector was transfected into HCT116 and SW480 cells. RT-QPCR was used to detect the effect of spl on the expression of TINCR. Co-transfected c-myc and spl expression vectors into HCT116 and SW480 cells, RT-qPCR was used to detect the expression of TINCR.Results1. Downregulation of LncRNA-TINCR correlated with CRC progression.(1) TINCR expression in CRC tissues.In oncomine database, TINCR was significantly downregulated in colorectal cancer tissues comparing with adjacent tissues. The expression of TINCR in tumour and paired non-tumour tissues obtained from 44 patients with CRC were detected by RT-qPCR, and the results revealed significantly lower TINCR expression in 38 of 44 CRC specimens (P<0.05). We next examined the relationship between TINCR expression levels and the clinicopathological characteristics of the tumour tissue samples. The rusults shown that TINCR expression level was reversely correlated to serosal invasion (p=0.001), lymph metastasis (p=0.037), and tumour node metastasis (TNM) classification (p=0.016), while positively correlated with differentiation degree (p=0.017). TINCR expression has no significant with patients’gender, age, and tumor size (P>0.05, P>0.05, P>0.05).(2) TINCR expression in CRC cells.The expression level of TINCR in the high malignant potential cell lines LoVo, RKO, and SW620 was significantly decreased compared with low malignant potential cell lines HCT116, SW480 and LS174T.(3) The location of TINCRThe transcript for TINCR was mainly located in the cytoplasm of HCT116 and SW480 cells by separating nuclear and cytoplasmic RNA fraction.2. TINCR inhibits CRC cells proliferation, migration and metastasis.(1) Successfully constructed the TINCR knockout cells in HCT116 and SW480, labelled as HCT116/shTINCR and SW480/shTINCR. Compared with the NC group, TINCR was significantly decreased in HCT116/shTINCR and SW480/shTINCR cells.Successfully constructed the TINCR knockin cells in RKO and LoVo, labelled as RKO/oeTINCR and LoVo/oeTINCR. Compared with the vector group, TINCR was significantly decreased in RKO/oeTINCR and LoVo/oeTINCR cells.(2) CCK-8 assay showed that the down regulated TINCR promote cell proliferation in HCT116/shTINCR and SW480/shTINCR cells (P<0.05, P<0.05); However, up regulated TINCR restrained the cell proliferative abilities in RKO/oeTINCR and LoVo/oeTINCR cells. The plate colony formation assays yielded the similar effect.(3) Transwell migration assays showed that down regulated TINCR markedly promoted the motility of HCT116/shTINCR and SW480/shTINCR cells (P<0.05, P <0.05), while up regulated TINCR restrained cell motility in RKO/oeTINCR and LoVo/oeTINCR cells.(4) Subcutaneous tumour growth in the HCT116/shTINCR was faster than the NC groups (P<0.05). Immunostaining confirmed that the cell proliferation index Ki-67 was increased in the HCT116/shTINCR.(5) Xenograft tumours generated by Tail vein injection showed that HCT116/shTINCR cells had more liver and lung metastasis ability compared with NC groups (P<0.05). All 5 mices had lung metastasis locis in HCT116/shTINCR group, while only 1 had in NC group.4/5 mices had liver metastasis locis in HCT116/shTINCR group, while no liver metastasis loci in NC group.3. TINCR specifically binds to EpCAM and regulates its proteolysis.(1) RT-qPCR, Western blot were used to detect the EpCAM mRNA and protein level in HCT116/shTINCR and SW480/shTINCR, and the results shown that the downregulation of TINCR did not change the EpCAM mRNA but downregulated the EpCAM protein levels in CRC cells.(2) The half-time of EpCAM in HCT116/shTINCR and SW480/shTINCR cells were detected by Western blot, the results shown that the knockdown of TINCR led to a robust decreased in EpCAM protein half-life in HCT116 and SW480 cells.(3) RNA immunoprecipitation assays demonstrated that EpCAM Ab precipitated the lncRNA-TINCR while the IgG Ab could not. Secondary, biotin RNA pull-down assay was performed, suggesting that TINCR directly interacted with EpCAM.(4) The overexpression of Psen2 alone or in HCT116/sh-TINCR cells upregulated the expression of EpICD and reduced the expression of EpCAM. However, all these effects disappeared when TINCR was re-expressed in HCT116 NC or sh-TINCR cells.(5) EpICD was upregulated in the CRC tissues compared with their normal mucous. We next analyzed the expression of EpICD in the same 44 paired CRC tissues by IHC. Spearman correlation analyses showed that TINCR and EpICD were inversely related in expression (p=0.002, r=-0.478).4. Downregulation of TINCR activates WNT/β-catenin pathway in CRC.(1) TOP/FOP assay indicated that the knockdown of TINCR resulted in 200%-300% increment of TOP-Flash reporter gene activity.(2) The expression of β-catenin, as well as the Wnt target genes TCF4, and c-Myc were significantly induced in TINCR-knockdown cells.(3) co-IP assay indicated that EpICD and β-catenin could form more complex in HCT116/shTINCR cells than NC group.5. c-Myc represses the expression of TINCR through inhibiting the transcriptive activity of Spl(1) Potential transcription factors (TFs) regulating the expression of TINCR were analyzed by bioinformatics algorithms. Spl was the predicted TF with one binding site on the TINCR promoter showed by the JARSPAR, and PROMO databases.(2) TINCR was markedly increased in HCT116 and SW480 cells when overexpressed sp1 protein. Dual luciferase report system assay indicated that sp1 could increase the luciferase activity in pGL3-wt group not pGL3-mut and pGL3-basic group. ChIP assays confirmed the Sp1 binding to TINCR promoter.(3) The overexpression of Sp1 significantly increases the expression of TINCR in HCT116 and SW480 cells. TINCR, transcribed by Sp1, was repressed by c-Myc. Moreover, the increasing expression of TINCR by Sp1 was partly suppressed by c-Myc.Conclusion1. TINCR was downregulated in colorectal cancer tissues. TINCR expression level was reversely correlated to serosal invasion, lymph metastasis, and tumour node metastasis (TNM) classification, while positively correlated with differentiation degree. The expression level of TINCR in the high malignant potential cell lines was significantly decreased compared with low malignant potential cell lines. All these indicated that TINCR function as a tumor suppress gene in CRC.2. TINCR inhibits CRC cells proliferation, migration and metastasis. In CRC, loss of TINCR promotes proliferation and metastasis.3. TINCR specifically binds to EpCAM and regulates its proteolysis. In CRC, down regulated TINCR promotes the cleavage of EpCAM, and subsequently release intracellular fragment EpICD.4. Downregulation of TINCR activates WNT/β-catenin pathway in CRC.5. c-Myc represses the expression of TINCR through inhibiting the transcriptive activity of Sp1.