VPS33B Can Suppress Cell Proliferation, Metastasis And Invasion In Human Lung Cancer

BackgroundPrimary bronchial lung cancer is the most common malignant tumors as well as the leading cause of death from cancer worldwide which derives from the primary bronchial mucosa and alveolar.The main reason of causing lung cancer is now recognized as smoking, environmental pollution and radon exposure.Lung cancer is the leading cause of cancer deaths in males world wide, and the mortality burden for lung cancer among females in developing countries was as high as the burden for cervical cancer. This trend is particularly evident in developing countries that more than one million patient died of lung cancer every year in the world and the mortality of lung cancer still increased at a speed of 1～5% per year. The incidence of lung cancer growing at a speed of 26.9% a year at present, with the continuent and rapid rising of lung cancer incidence and mortality in China. The majority of lung cancers are diagnosed at a distant stage because early disease is typically asymptomatic, patients with lung cancer often exhibit tumor cell invasion and metastasis before diagnosis and treat. Hence, despite of several years of continuous research the pathogenesis of lung cancer, methods now have produced a variety of treatment of lung cancer, but improving survival in lung cancer is a major challenge for modern oncology considering that 5-year survival remains<15%. Histologically, lung cancer is classified into two broad categories; small-cell lung cancer (SCLC), occurring in approximately 15% of patients and the more prevalent NSCLC, which accounts for approximately 85% of cases.Current the treatments of NSCLC main including surgery, radiotherapy, chemotherapy. Biological treatment is cared about with the development of the research on the deepening of NSCLC.With the further development of molecular biological research promote the drive cancer gene mutation found.VPS33B official name is Vacuolar protein sorting 33 homologB(yeast), which expression protein called Vacuolar protein sorting-associated protein 33B, belong to Secl/Munc-18 (SM) protein family member, the human ortholog of rat Vps33b. VPS33B encodes a transcript of 2482 nt with an ORF of 617 amino acids and a predicted protein size of 70.6 kDa. VPS33B contains a Sec-1 domain shared with a family of proteins involved in protein sorting and vesicular trafficking, classifying small molecules of intracellular transport to the corresponding target organ to make its function in the body.Current reports of VPS33B gene mainly concentrated in congenital joint contractures, renal tubular dysfunction, and Cholestasis(ARC syndrome) and megakaryocyte and platelet alpha-granule biogenesis. We haven’t seen the related report about its relationships with tumour development.Objective1. To identify VPS33B expression features in lung cancer cells and in lung tissues respectively;2. To make sure the role of VPS33B in regulation of lung cancer cell proliferation, and then to investigate the molecular mechanism of the role;3. To make sure the role of VPS33B in regulation of lung cancer cell migration and invasion and then to investigate the molecular mechanism of the role.Contents and methods1. To identify VPS33B expression features in lung cancer cells and in lung tissues respectivelymRNA and Protein expression of VPS33B in lung cancer tissues and noncancerous lung tissues were measured by Real-time RT-PCR and immunohistochemistry; protein levels of VPS33B expression were detected by Western blot in lung cancer cell lines.2. To make sure the role of VPS33B on lung cancer cell proliferation and identify the molecular mechanism in lung cancer in vitro1) Use of packaging lentivirus constracted by company to transfect A549 and H1975, establishing cell lines with stable expression VPS33B;transfection synthetic siRNA targeting VPS33B gene into lung cancer cells with VPS33B overexpression; Fluorescence quantitative PCR and Western blot were used to identify the overexpress efficiency and interference efficiency, the best interference effect among the three interference sequences be used for further experiment;2) Activity pf lung cancer cell lines were detected by MTT after overexpressing and silencing expression of VPS33B;3) Capacities of proliferation in lung cancer cell lines were detected by plate colony formation assay after overexpressing and silencing expression of VPS33B;4) Percentage of cells in S phase of cell cycle in A549 and H1975 after overexpressing and silencing expression of VPS33B was detected by EDU and cell cycle analysis;5) The effect of the capacity of tumor-forming was detected by the test of formation of tumor in nude mice in vivo after VPS33B overexpressed, and then got out of the tumor, implemented tissue section and immunohistochemical detection, then observed the changes of expression quantity of related factor;6) The change of some important proteins related cell cycle in A549 and H1975 after overexpressing and silencing expression of VPS33B was detected by Western blot and the related molecular mechanism was also studied primary;3. To investigate the role of VPS33B on lung cancer cell metastasis and invasion and identify the related molecular mechanism in lung cancer in vitro1) The abilities of metastasis and invasion in lung cancer cell lines were detected by Transwell and Boyden test after overexpressing and silencing expression of VPS33B;2) The change of some important proteins related EMT in A549 and H1975 after overexpressing and silencing expression of VPS33B was detected by Western blot and got a primary knowledge about the related signal pathways.Results1. Expression of VPS33B in lung cancer and its clinical significanceUsing Real-time RT-PCR, we found that comparing to noncancerous lung tissues, VPS33B expression was down-regulated in lung cancer. Immunohistochemistry showed that VPS33B protein was mainly localized in cytoplasm and significantly down-regulated in lung cancer comparing with noncancerous lung tissues. The expression of VPS33B protein closely related to clinical stage of lung caner patients: the clinical stage sooner, VPS33B express stronger; while the later clinical stage, VPS33B express weaker or negative.2. VPS33B suppressing cell proliferation in lung cancer and its molecular mechanism1) Establishment of the stable lung cancer cells of VPS33B overexpressionAfter VPS33B was introduced into A549 and H1975 lung cancer cells, we established stable VPS33B overexpressed lung caner cells, which were chosen for further function and molecular mechanism study;2) A preliminary study of the function and molecular mechanisms after VPS33B overexpression in lung cancerCompared to A549-NC and H1975-NC negative control cells, both A549-VPS33B and H1975-VPS33B cells showed to be inhibited significantly in MTT cell proliferation assay, plate colony formation assay. Furthermore, cell cycle analysis showed that the G1 phase increased significantly and S phase decreased markedly in A549-VPS33B and H1975-VPS33B cells in comparison to A549-NC and H1975-NC negative control cells;3) The VPS33B overexpressed lung cancer cells and control cells were inoculated subcutaneously in nude mice. After two or three weeks, we found VPS33B overexpression significantly inhibited the proliferation of lung cancer cells in vivo.4) Western blot analysis was used to test the change of several important proteins associated with cell cycle progression and other important signaling pathway proteins before and after VPS33B overexpression. The results showed that the expression of C-myc, CDK6, CCND1, p-Rb and E2F1 in A549-VPS33B and H1975-VPS33B cells was down-regulated, while p 16 expression was up-regulated. Western blot also showed that the expression of p-AKT and p-PI3K was down-regulated,but AKT, PTEN and PI3K had no significant change.3. VPS33B suppressing cell metastasis and invasion in lung cancer and its molecular mechanism1) Transwell and Boyden test showed that capacities of metastasis and invasion in lung cancer cell lines were suppressed after overexpressing VPS33B;2) Western blot showed that some proteins related EMT such an N-Ca, Vimentin were downregulated whereas E-Ca were upregulated in A549 and H1975 after overexpressing VPS33B.4. Effects of Inhibition VPS33B expression on the biological function of lung cancer cellsChemically synthesized siRNA was transfected into lung cancer cells A549-VPS33B and H1975-VPS33B by cationic polymer transfection reagent, and then real-time RT-PCR and Western blot were applied to detect silence effects after 48h or 72h. The results showed that the three interference sequences had the same interference effect, and each one can be used for further experiment. In MTT cell proliferation assay and cell cycle analysis, we found that in comparison to A549-VPS33B/Si-NC and H1975-VPS33B/Si-NC negative control cells, the proliferation ability of A549-VPS33B/Si-VPS33B and H1975-VPS33B/Si-VPS33B cells was markedly increased. The ability of metastasis and invasion also had the significant change.Conclusions1.VPS33B gene is closely associated with lung cancer development;2. VPS33B can suppresses the proliferation ability of lung cancer cells;3. VPS33B gene can suppresses the metastasis and invasion ability of lung cancer cells.