MicroRNA-582-5p Targeting Inhibition Of Rab27a-mediated Colon Cancer Cells And Its Molecular Mechanism

Research objective:Carcinoma of colon (or colon cancer, CRC) has become a cancer with high incidence and high mortality rate, however, the underlying molecular mechanism is not clear and there is no uniform understanding. MicroRNA has been widely reported to play important roles in the regulation of the cell function of CRC. One of the important members of miRNAs, miR-582-5p, has been showed a low level in CRC cells. Suggesting that it may be involved in the regulation of CRC. This paper aims to study the role of miR-582-5p in mediated CRC and to explore the internal mechanism. In the first place, the studies of the expression of miR-582-5p in 25 cases of patients’ CRC tissues with colon cancer and human CRC cell lines (SW480, HCT-116, LoVo, HCT8, and SW620) were performed and the effects of miR-582-5p in CRC cell proliferation, apoptosis, invasion and cell cycle were analysed. And the regulation function of miR-582-5p on CRC occurrence and development were studied. Secondly, MiR-582-5p targeting transcription factor Rab27a regulates the proliferation, invasion and cell cycle of CRC cells were studied. Finally, in order to find the molecular mechanism of miR-582-5p targeting Rab27a mediated regulation of CRC, the regulation of miR-582-5p for detection of Rab27a related signaling pathways was studied.Methods:1. The change of miR-582-5p level in CRC tissues, paracancerous tissues (NCT) and CRC cell lines were tested by using qRT-PCR. The cells were transiently transfected by miR-582-5p mimic and overexpressed miR-582-5p. The use of Western blot and cell viability assay (MTT), cell cycle analysis, apoptosis assay and cell invasion assay from the molecular and cellular level were performed to study the effects of miR-582-5p in colon cancer cells.2. The use of bioinformatics and luciferase reporter assay were performed to screen and confirm the direct target gene of miR-582-5p in the regulation of CRC.3. Construction of Rab27a overexpression vector, miR-582-5p mimic-transfected SW480 and HCT-116 cells were further transfected with Rab27a overexpression plasmids. And qRT-PCR, Western blot, MTT assay, cell cycle analysis, apoptosis assay and invasion were used to validated miR-582-5p targeting transcription factor Rab27a regulating CRC.4. Construction of FAK siRNA lentiviral slience expression vector and the use of Integrin inhibitor Cilengitide (CIL), with the help of qRT-PCR and Western blot experiments to explore the relationship between miR-582-5p negative regulation of Rab27a and the regulation of Integrin signal transduction pathway in colon cancer cells. And qRT-PCR, Western blot, MTT assay, cell cycle analysis, apoptosis assay and invasion were used to explore the effects of Integrin signal transduction pathway in miR-582-5p/Rab27a axis mediated CRC cell motility, invasion, apoptosis and cell cycle. Detection of Rab27a, Integrin-β1, FAK and p-FAK (Y397) expression were performed in clinical tissue of CRC.Results:1. The low level of miR-582-5p were tested in normal colon, stomach and small intestine gastrointestinal system, liver and spleen and other digestive tissues. The further detection found that the miR-582-5p expression in 19 cases colon tissues of patients in 25 patients with CRC was significantly decreased. And the expression of miR-582-5p in CRC cells (HCT8, HCT-116, LoVo, SW480 and SW620) and CRC tissues were decreased compared with the control group and there were no changes in tumor adjacent tissues. miR-582-5p through the inhibition of cell cycle G1 to S development and restricts the proliferation of CRC cells:(1) MTT cell activity assay showed that the overexpression of miR-582-5p inhibits the proliferation of CRC cells. (2) The population of CRC cells transfected miR-582-5p mimic were significantly increased in the G1 phase, but the decreased effects were shown in the S phase. The overexpression of miR-582-5p could promote and increased the apoptosis of HCT-116 and SW480 cells. (3) Western blot analysis of Rb/E2F1 expression showed that Rb was shown a low level but E2F1 was shown a high level in CRC lines, overexpression of miR-582-5p could increase the expression of Rb and decrease the E2F1 expression. Transwell cell invasion experiments showed that overexpression of miR-582-5p could inhibit the invasion of CRC cells. PARP-1 in CRC cell lines was down regulated by overexpression of miR-582-5p, and PARP-1-cleaved form was overexpressed by miR-582-5p mimic. And overexpression of miR-582-5p inhibits the expression of mTOR and its related signal molecules of Akt, pAk, ERK1/2 and pErkl/2.2. In CRC cell lines, qRT-PCR and Western blot detection showed that overexpression of miR-582-5p significantly inhibited the expression of Rab27a. The results of bioinformatics prediction and luciferase reporter experiment showed that Rab27a is a directed target gene of miR-582-5p in the regulation of the CRC pathological process.3. The stable over expression vector of Rab27a were constructed successfully, miR-582-5p mimic-transfected SW480 and HCT-116 cells were further transfected with Rab27a overexpression plasmids. The experimental results of MTT, cell cycle experiment and invasion experiments showed that Rab27a overexpresson could reverse the effecs of miR-582-5p in the inhibition of cancer cell proliferation and invasion. The overexpression of Rab27a also reduce the inhibition effects on E2F1, PARP-1, ERK1/2, p-ERKl/2 and PI3K/Akt pathway related protein which induced by miR-582-5p mimic; the upregulation of Rb and PARP-1 cleaved form induced by miR-582-5p mimic has been reversed by Rab27a overexpression.4. MiR-582-5p mimic-transfected SW480 and HCT-116 cells were further transfected with Rab27a overexpression plasmids. The test of the key protein of Integrin signal transduction pathway which included Integrin-β1、FAK、p-FAK(Y397) and the downstream moleculars including Wnt signaling and Ras/raf/ERK pathway were performed by using Western blot. The results showed that The overexpression of Rab27a also reduce the inhibition effects on Integrin-β1,FAK, p-FAK(Y397) and the key protein of Wnt signaling and Ras/raf/ERK pathway which induced by miR-582-5p mimic.The Integrin signal transduction pathway inhibitor Cilengitide (CIL) could downregulate Rab27a, BAX, BAK, IGF-II, ILK, cyclin D1 and the key protein of Wnt signaling and Ras/raf/ERK pathway. The siRNA lentiviral expression vector of FAK transfected into SW480 and HCT-116 showed similar effects with CIL. In 25 patients CRC tissues we obtained in 19 cases which the expression of miR-582-5p was downregulated (miR-582-5p-D), and 6 cases of miR-582-5p was not downregulated (miR-582-5p-ND). The results showed that the expression of Rab27a, Integrin-β1, FAK and p-FAK (Y397) in CRC tissues were all upregulated, and a careful comparison in the results, we found that the increase rate of Rab27a, Integrin-P 1, FAK and p-FAK(Y397) in miR-582-5p-D tissues were more significant than miR-582-5p-ND tissues.ConclusionIt was demonstrated that miR-582-5p was downregulated in CRC tissues and cell lines. Overexpression of miR-582-5p inhibited CRC cell growth, cell cycle progression and invasion, as well as increased cell apoptosis by targeting Rab27a. The Integrin signal transduction pathway and FAK mediated regulation of miR-582-5p is mainly targeting Rab27a inhibition of cancer cell behavior factors. FAK mediated Integrin signal transduction pathway might be the mean way that miR-582-5p inhibits of CRC cells behavior by targeting Rab27a. This research might provide a therapeutic strategy for patients with CRC.