Rackl Knockdown Suppresses Cell Proliferation And Promotes Epithelial-Mesenchymal Transition In A549Cells

BackgroundThe receptor for activated C kinase1(Rack1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins. Rack1adopts a seven-bladed β-propeller structure which facilitates protein binding and allow Rackl to interact with numerous proteins. Rackl is the scaffolding protein for many kinases and cellular shuttling proteins. As a result, Rackl is a key mediator of various pathways and contributes to numerous aspects of cellular function, involving cell proliferation, cell migration, angiogenesis, transcription and translation. Moreover, there exists aberration of Rack1expression in angiogenesis and many human tumors. Although it is largely reported that Rackl plays an important role in multiple tumors through different mechanisms, the role of Rack1in NSCLC needs to be defined.During the past decades, the incidence and mortality of lung cancer ascend straightly worldwide, especially in industrial developed countries. Lung cancer, the leading cause of cancer deaths worldwide, falls into two categories:small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC is epithelium-derived lung cancer other than small cell lung cancer, including squamous carcinoma, adenocarcinoma and large cell carcinoma. NSCLC is the most common form of lung cancer, and makes up80-85%of the lung cancer patients. One characteristic of NSCLC is slow progression and hard to make early diagnosis, thus most of the patients are not suitable to take surgery after diagnosis. It is necessary to make extensive research on the molecular mechanism of the genesis and progression of NSCLC, and look for new early prognostic markers, predictors.A549is a cell line derived from NSCLC, commonly used in research on NSCLC and epithelial to mesenchymal transition (EMT).ObjectivesThe project are supposed to investigate the effect of Rack1on the genesis and progression of NSCLC and the underlying molecular mechanism, and promote the clinical application of Rack1as one of markers for early diagnosis and prognosis prediction.Methods1. K-rasG12/D was activated via the trachea instillation of Adeno-Cre to induce the genesis of pulmonary carcinoma in mice. The expression of Rackl was examined in the existent mouse model with the KrasG12/D-induced lung cancer.2. Via shRNA lentivirus infection system, we established the cell lines with Rack1knockdown.3. The cellular proliferation, colony formation ability, migration capacity were detected by MTS assay, soft agar colony formation assay, and wound healing assay, respectively.4. In order to investigate the potential molecular mechanism of this phenomenon, we examined the expression level of some key molecules in EMT via western blot.Results1. We found that the expression level of Rackl was upregulated in lung adeno carcinoma of mouse, compared to that of normal tissue.2. Two cell lines with Rackl knockdown and control cell line (shMock) were successfully established. The expression of Rackl was downregulated to46%(shRackl-1) and44%(shRack1-2), respectively.3. The knockdown of Rack1in A549cells suppressed cell proliferation and the ability of colony formation in soft agar, promoted the cell migration, and made cell vulnerable to EMT.4. Rack1knockdown downregulated the expression of E-cadherin, enhanced phosphorylation level of extracellular signal-regulated kinase (ERK), increased the stability of c-Jun, and elevated activator protein-1(AP-1) activity.