Investigation Of Role For TSPAN12 In Chemoresistance And Proliferation Of SCLC Under Regulation Of MiR-495

IntroductionRecent reports revealed the fact that lung cancer is one of the most prevalent malignant tumors and the leading cause of cancer mortality in the world, furthermore, its incidence trends to raise year by year. The situation is even more severe in China, namely, as statistics from National Health and Family Planning Commission updated in December 2014 showed, the morbidity and mortality of lung cancer have pushed into first place with a rapid growth, leading to great harms to people. Primary lung cancer can be classified as non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and the later one represents around 20%. Multiple genes may involve in the development of SCLC, including oncogenes like Bcl-2, myc, ras and c-erbB-2, as well as tumor suppressor genes such as p53, RB gene, CDKN2 and FHIT, et cetera. Besides, it is reported that PI3K/AKT/mTOR signal pathway as well as methylation of caspase-8 (an important anti-apoptosis gene) is also involved in the process of SCLC. It is generally acknowledged that the cancer cell may originate from Kulchitsky cell, which is characterized by its production of ectopic hormone like L-dopa decarboxylase, bombesin and enzyme neuron-specific enolase. SCLC is aggressive with a high cell mitotic index and early metastasis (approximately two thirds of patients with SCLC are defined as metastatic ones). Although SCLC is sensitive to both chemotherapy and radiotherapy, it is characteristic of high relapse rate and poor prognosis due to acquired drug resistance. There are embers of hope for new therapeutics, thus, breaking chemoresistance of cancer cell maybe one of the main goals for SCLC treatment.Tetraspanins, belonging to the transmembrane 4 superfamily (TM4SF), are phylogenetically ancient transmembrane proteins with four characteristic hydrophobic domains. Study on tetraspanins is relatively recent and therefore there is much to learn about. So far, there are 33 tetraspanins are found in human, which are generally thought to typically anchor multiple proteins, such as integrin, growth factor and its receptor, human leukocyte antigen as well as major histocompatibility complex, to form tetraspanin-enriched microdomains (TEMs) as signaling platforms, affecting cancer cell proliferation, migration and adhesion. Besides, it is reported that tetraspanins contribute to cancer metastasis or viral infection, as well. Transmembrane 4 superfamily member 12 (TSPAN12, also NET-2), a member in TM4SF, plays a vital role in retinal vascular development and the depletion or mutation of this gene could subsequently results in familial exudative vitreoretinopathy (FEVR). Otherwise, TSPAN12 is reported to be one of the 33 tetraspanins that play important roles in the course of human tumor progression. Wachi S reported that microarrays of lung, breast and prostate cancer show significantly altered TSPAN12 expression. Knoblich K found that TSPAN12 can promote the proliferation of breast cancer while suppress cancer cell migration via P-catenin. Moreover, data from Otomo R revealed that p53 mutation in stroma cell could up-regulated the expression of TSPAN12 and promote the proliferation, invasiveness and migration of adjacent cancer cell. TSPAN12 seems to be a promoter in cancer progression. However, investigation on role for TSPAN12 in SCLC has not been reported yet. A cDNA microarray analysis performed in cellular models of SCLC, which are widely used as sensitive (H69) and resistant cell lines (H69AR) to chemotherapy, revealed that gene TSPAN12 was over-expressed in H69AR comparing with H69, suggesting that TSPAN12 may participate in process of SCLC chemoresistance. To research into function for TSPAN12 on the proliferation and chemosensitivity of cancer cell may help us to understand molecular features of SCLC and lead to new drug targets.MicroRNA (miRNA) is an evolutionarily ancient and single stranded small non-coding RNA with 20 to 24 nucleotides, which expresses in a pattern of spatial and temporal restriction during the development of diverse living organisms, including humans. Matured miRNA functions in RNA silencing and post-transcriptional regulation of gene expression via an active RNA-induced silencing complex (RISC) that base-pairing with complementary sequences within mRNA molecules following a series of gene transcription process. As a result, these mRNA molecules are silenced by one or more of the following processes in terms of their sequence similarity:1) cleavage of the mRNA strand into two pieces,2) destabilization of the mRNA through shortening of its poly (A) tail, and 3) less efficient translation of the mRNA into proteins by ribosomes, and then contribute to biological processes involving in early embryonic development, cell proliferation, apoptosis and cell differentiation as well. Bioinformatics prediction reveals that a given miRNA may have hundreds of different mRNA targets, and a given target mRNA might be regulated by multiple miRNAs. Just as miRNAs almost involve in every step of cell development or biological processes, so has the dysregulation of miRNAs been associated with disease. Various types of cancer have been found to be relative to miRNA deregulation, and accordingly "oncomirs" have been introduced and serve as oncogenes or tumor suppressors, for instance, bantam-miRNA is the first to be recognized as one. Besides, miRNAs also play roles in tumorigenesis via their target mRNAs related which, like miR-15b targeting MTSS1, miR-199a-5p targeting FZD6, miR-126 targeting EGFL7 as well as miR-200b targeting ZEB2, are crucial to breast cancer, colorectal cancer and lung cancer, respectively. Among all the miRNAs, miR-495, mapped to the 14q32.31 locus, is one of the regulators of embryonic development and a member of "oncomirs" by regulating differentiation of stem cells as well as the proliferation, migration, invasion of tumor cells. And investigations on miR-495 indicated it also important for cancer cell drug susceptibility. Song L reported that miR-495 can affect response of NSCLC to platinum by targeting ATP7A. When comes to leukemia, Xu Y showed that MDR1 may be involved in cancer cell multidrug resistance under regulation of miR-495. Additionally, it was reported that miR-495 could inversely regulate Etk/BMX-induced epithelial-mesenchymal transition and take part in the course of SCLC anti-cancer drug resistance. By microRNA array, miR-495 was down-regulated in anti-cancer drug resistant cells when comparing with chemosensitive clones. Moreover, gene sequence of TSPAN12 was partially complementary to that of miR-495. Taking evidences above into consideration, we hypothesized that miR-495 may regulate the drug susceptibility of small cell lung cancer via interaction with TSPAN12.The Wnt signaling pathways are a group of signal transduction pathways of highly evolutionarily conservation across many species. The canonical Wnt signaling pathway involves in a diverse family of secreted Wnt proteins, a Frizzled family receptor, Dishevelled protein, β-catenin protein, β-catenin destruction complex (including the following proteins:APC, GSK3β, Axin and P-catenin) and the TCF/LEF transcription family. As soon as Wnt binds Frizzled and LRP5/6 proteins, a signal is sent to the phosphoprotein Dsh and then the β-catenin destruction complex function becomes disrupted, leading to accumulation and subsequent activation of β-catenin, which stimulates the TCF/LEF-mediated transcriptional programs. As was reported, the Wnt signaling pathways function for embryonic early development, the formation of important tissues, tissue regeneration and epithelial mesenchymal transition (EMT) as well. However, it was also reported that the activation of P-catenin could be wholly independent of Wnt protein during the course of retinal vascularization, in which P-catenin signaling pathway was activated by directly interaction of TSPAN12 and FZD4. The dysfunction of Wnt/β-catenin signaling pathway was recognized as guide to tumor. And in the investigation of effect of TSPAN12 on the cell proliferation of breast cancer, Knoblich K found that β-catenin signaling pathway was of function. Norrin/p-catenin signaling pathway may play a role in the process of tumor progression. The result from the cDNA array of SCLC revealed that TSPAN12 regulate the cell proliferation and the formation of drug resistance in small cell lung cancer. We then hypothesized that TSPAN12 may also function for SCLC via Norrin/β-catenin signaling pathway.ObjectiveBy microarray, we found out significantly altered expression genes between SCLC anti-cancer drug resistant cell, H69AR, and its parental clone H69. Then we chose remarkable over-expressed TSPAN12 for further study, analyzing its function on the proliferation and drug susceptibility of cancer cell as well as the possible mechanisms, which may help to clue in clinical therapy.Content1. To compare the expression of TSPAN12 in SCLC cancer cell H69AR to H69, H446/CDDP and H446 were selected to verify the result.2. To detect the influence of TSPAN12 on the drug susceptibility and proliferation of SCLC cancer cells.3. To detect the different expression of miR-495 from H69AR to H69 and analyze its potential regulation to TSPAN12.4. To analyze the effect of TSPAN 12 on the activation of P-catenin signaling pathway.Methods1. Through gene chip, we detected a significantly changed TSPAN 12 expression in SCLC cancer cells between H69AR and H69. We further verified the result by qRT-PCR and western blotting in H69 series and H446 series.2. By CCK-8 assay, we analyzed the change response in cancer cells to DDP and VP-16 after transfection of shRNA. Meanwhile, we compared the proliferation of cancer cells before and after treatment through CCK-8 assay and clone formation assay. Flow cytometry was used to detect cell apoptosis and cell cycle.3. We found that miR-495, whose expression was expressively altered in microRNA arrays of H69AR and H69, may be a regulator of TSPAN 12 according to data from microRNA.org. By qRT-PCR, we affirmed the result in both cell series.4. We further established has-miR-495 mimics and has-miR-495 inhibitors to up-or down-regulated miR-495 expression in SCLC cells and subsequently detected the TSPAN12 on the level of mRNA and protein.5. To understand the effect of TSPAN12 on the activation of β-catenin signaling pathway, we detected the expression of β-catenin in SCLC cancer cells before and after shRNA treatment via qRT-PCR and western blotting. Moreover, we detected the distribution of β-catenin in cells through immunofluorescence assay.Statistical analysisThe experiments were repeated independently at least three times. Data were represented as Mean±SD values and all statistical analyses were processed through SPSS 13.0 software. Difference in expression of TSPAN12 or miR-495 between chemoresistant cells and chemosensitive cells were evaluated by independent-samples T test, while data of cell cycle and apoptosis among groups were analyzed through one-way ANOVA or Welch test. The initial F test was followed up with Dunnett or Dunnett’s T3 multiple comparisons procedure. Results of CCK-8 experiment were by One-way analysis of variance with SNK multiple comparisons procedure. P< 0.05 values were considered to indicate statistically significant.Results1. By cDNA microarray, we detected that TSPAN12 was 3.15-fold over-expressed in H69AR comparing with that of H69, which was consistent with results of qRT-PCR (4.66-fold, P=0.033) and western blotting (1.5-fold, P=0.01). Experiments on another cell series H446 and H446/CDDP got uniform outcomes. 2. Cytolysis of H446/CDDP, transfected with TSPAN12 shRNA or not, was separately analyzed by qRT-PCR and western blotting, which showed that the expression of TSPAN12 mRNA decreased to 28.667% and 65.333%in cells with transfection of TSPAN12-1082 and TSPAN12-748, respectively. We then chose H446/CDDP cells with TSPAN12-1082 for further study.3. SCLC chemoresistant cells become less resistant to DDP and VP-16 after treatment with shRNA TSPAN12-1082 in CCK-8 assay, in which matching IC50 decreased accordingly. However, data were with no significant difference following statistical analysis.4. In the experiment of proliferation, we got a limit velocity to cell multiplication (P <0.05) and an impaired capacity for cloning (P=0.022) in H446/CDDP cells with depletion of TSPAN12 detected by CCK-8 assay and clone formation assay, respectively.5. By flow cytometry, we detected a G0/G1 arrest (P=0.01) and an increased DDP-induced cell apoptosis (P=0.002) in TSPAN12-depleted H446/CDDP, versus H446/CDDP.6. In microRNA array, we found that the expression of miR-495 in H69AR was markedly suppressed comparing H69, which was also verified through qRT-PCR in both two cell series.7. We found it the-22-nucleotide RNA miR-495 contained sequences partially complementary to multiple sequences in the 3’UTR of the TSPAN12 mRNA. And when expression of miR-495 in H69AR was up-regulated to 3.31-fold with miR-495 mimics, expression of TSPAN12 was decreased on level of mRNA (0.67-fold, P=0.045) and protein (0.57-fold, P<0.001). And when H69 was treated with miR-495 inhibitors, expression of miR-495 decreased to 0.42-fold, while that of TSPAN12 increased to 4.76-fold (P=0.018) and 2.58-fold (P<0.001) detected by qRT-PCR and western blotting, respectively. Experiments repeated in H446 series got similar results.8. By qRT-PCR, we detected the mRNA expression of β-catenin, MYC and Cyclin D1 in depleted-TSPAN12 H446/CDDP cells decreased to 47%,48% and 38%(P <0.05) versus that of H446, respectively. And on protein level, P-catenin was 72% down-regulated in transfectants. The results were verified in the assays of H69 series. Moreover, depletion of TSPAN12 in cancer cells could restraint β-catenin migration to cell nucleus showed in immunofluorescence assay.Conclusion1. The gene TSPAN12 was over-expressed in SCLC chemoresistant cancer cell H69AR and depletion of TSPAN12 in cell showed greater sensitivity to anti-cancer drugs while suppressed its proliferation, suggesting TSPAN12 could be a promoter in SCLC progression, but not an essential factor for SCLC chemoresistance devlopment.2. TSPAN12 could affect cancer cell cycle and drug-induced apoptosis.3. MiR-495 was reduced in H69AR comparing to H69 and increase or decrease of its expression could down-or up-regulate the expression of TSPAN12, conversely, indicating TSPAN12 may be a target of miR-495.4. Depletion of TSPAN12 in cancer cells could decrease the expression of P-catenin and restraint its migration to cell nucleus, suggesting TSPAN12 may function on SCLC via β-catenin signaling pathway.