MicroRNA-335 targets the MEK/ERK pathway to regulate the proliferation and metastasis of colon cancer
Authors: Minghua Wang
Chuang Yang
Qiang Chi
Junde Zhou
Journal: American journal of translational research (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Colon cancer; TUNNEL assay; apoptosis; expression; invasion
Affiliations: Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University Harbin 150086, China.

Abstract:  Several studies have proved the tumor-suppressive effects of miR-335 but its role in colon cancer via regulation of the Raf/MEK/ERK signalling pathway is yet unknown. As such the main motive of conducting the present study was to elucidate the role of miR-335 in colon cancer via regulation of Raf/MEK/ERK signalling pathway and to explore its therapeutic potential. The results revealed significant (P &lt; 0.05) downregulation of miR-335 in colon cancer and its overexpression led to a significant (P &lt; 0.05) decline in viability of the HT-29 and SW948 cells. The TUNNEL assay showed miR-335 promotes apoptosis in the HT-29 and SW948 colon cancer cells and is also associated with increase in Bax and decrease in Bcl-2 expression. The results also revealed that miR-335 overexpression enhances the sensitivity of the HT-29 and SW948 cells to the apoptotic effects of cisplatin. From the transwell assays, it was found that the migration of the HT-29 and SW948 cells was decreased by 53% and 45% and while as invasion was decreased by 49% and 42% respectively (P &lt; 0.05). Finally, western blot analysis showed that miR-335 blocks the Raf/MEK/ERK signalling pathway in HT-29 colon cancer cells. The results of in vivo study showed that miR-335 also exhibits tumor-suppressive effects on xenografted tumors. Taken together, it is concluded that miR-335 acts as tumor-suppressor in colon cancer and may exhibit therapeutic implications in its treatment. 

Retracted: No

PubPeer comments:
  The RNA was extracted from the transfected HT-29 and SW948 cells using a Trizol regent and then the RevertAid cDNA synthesis kit was employed to synthesize complementary DNA (cDNA). The relative expression was evaluated through quantitative real time polymerase chain reaction (qRT-PCR). The 20 u00b5l PCR reaction mixture consisted of dNTPs (200 u00b5M) MgCl2 (1.5 mM), Taq DNA Polymerase (2.5 units), primers (0.2 u03bcM each) and cDNA (0.5 u00b5g). The parameters of PCR cycling were: 95u00b0C for 30 sec, followed by 40 cycles of 95u00b0C for 15 sec, and 58u00b0C for 30 sec. U6 snRNA and GAPDH were used as internal controls and u0394u0394CT methodology used for relative quantification as previously described [12]. The reference is to  12. Hua F, Li CH, Chen XG, Liu XP. Daidzein exerts anticancer activity towards SKOV3 human ovarian cancer cells by inducing apoptosis and cell cycle arrest and inhibiting the Raf MEK ERK cascade. Int J Mol Med. 2018;41:3485u20133492. The authors are under the impression that [Hua et al (2018)] describe the use of PCR to quantify targetted RNA. In fact they do not mention those topics. The same misapprehension occurred in [Zhong et al (2019)] , where we read  RNA was isolated from the tissues and cell lines using RNeasy kits, and cDNA was synthesized with an Omniscript RT (Qiagen, Inc.) kit from 2 u00b5g of the RNA. The cycling conditions were as follows: 95u00b0C for 20 s, followed by 40 cycles of 95u00b0C for 15 s, and 58u00b0C for 1 min. The expression was estimated by 2u2013DDCt method and actin was used as an internal control [13,14] - [14] being Hua et al. ------------------------------------------------ The influence of [Hua et al (2018)] is surprisingly far-reaching. Google Scholar informs us that their in-vitro study of Daidzein has been cited **71** times. Many of these are review papers, but we also find a surprising number of papers using it in  Apoptosis Assay paragraphs to illustrate the use of flow-cytometry and Annexin V PI staining to quantify cell apoptosis. Indeed, Hua et al did apply the method, but they were not pioneers, nor is their explanation superior to many others. Alternatively, the paper is cited as a precedent for a phytochemical (daidzein) inhibiting cancer proliferation by interrupting the cell cycle. Given all the earlier precedents, the choice again invites some explanation. It is almost as if all these papers were prepared by a third-party service (a papermill ), following a template from one paper to the next, without checking its contents. Consistent with this notion, the papers came to my attention by recycling the same figures (despite a lack of authors in common). - [Jia et al (2019)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR3 cells was performed as described previously [17]. - [Luo et al (2019)] .  Many plant-derived molecules have been reported to interfere with the **ROS** level in the cancer cells [13]. ... The slides were then covered with a covers lip and examined with a fluorescent microscope. Then 4u2019,6-diamidino-2-phenylindole (DAPI) and annexin V propidium iodide (PI) staining was performed as described previously [13]. - [Zeng et al (2019)] .  The slides were then covered with a covers lip and examined with a fluorescent microscope (BDBiosciences, San Jose, CA, USA). The annexin V PI staining was performed as described previously [8]. - [Wu et al (2019)] *[retracted]*,  The slides were covered with a coverslip and examined with a fluorescent microscope (BD Biosciences, San Jose, CA, USA). Annexin V PI staining was performed as described previously [13]. - [Yang, Chen Wang (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as described previously 16 . - [Yang et al (2019)] .  The slides were covered with cover slips and examined with a fuorescent microscope. DAPI and annexin V propidium iodide (PI) staining were performed as described previously [14] *[14 is a misprint for 13]*. - [Huang et al (2019)] .  The slides were covered with cover slip and examined under fluorescence microscope. Annexin V propidium iodide (PI) staining of the Icariin-treated HeLa cells was performed as described previously [18]. - [Liu et al (2019)] .  The distribution of the breast cancer SK-BR-3 cells in different cycle phases was performed by flow cytometry after PI staining, following the method reported by Hua et al [11]. ...After overnight incubation at 4u00b0C, the cells were subjected to PI staining and flow cytometry. - [Liu et al (2019)] .  After covering the glass slides with cover slips, the cells were analyzed under a fluorescent microscope (Nikon Instruments, Inc., NY, USA). For determining percentage of apoptotic cells, Annexin V PI staining assay using a FACSCalibur flow cytometry (BD Biosciences, NY, USA) was carried out using a previously reported method [12] - [Yin et al (2019)] .  The slides were covered with covers slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as previously described [13] . - [Zhang et al (2019)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining of the SW48 cells was performed as described previously [11] . - [Ma et al (2019)] .  The slides were cover-slipped and examined under a fluorescent microscope. Annexin V PI staining was performed as described previously [15]. - [Li et al (2019)] .  The slides were cover-slipped and assessed by fluorescent microscopy. ROS estimation was performed as described previously [13]. - [Zhang et al (2019)] .  The slides were covered with a cover slip and examined with a fluorescent microscope. Annexin V PI staining was performed as previously described [12]. - [Wu et al (2020)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the AGS cells was performed as described previously [15]. - [Yu et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [9]. - [Yin et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [10]. - [Wei et al (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR-3 cells was performed as described previously [11]. - [Wen et al (2020)] .  The glass slides laden with cell cultures were then covered with cover slips and then using a fluorescence microscope (BioTek Instruments, Inc., Winooski, VT, USA,) the cells were analysed. Quantification of apoptosis was done with annexin V PI staining assay which was performed as reported already (Hua et al., 2018). -------------------------------------------------------------- Cell-cycle arrest: - [Li Han (2019)] .  Cell cycle arrest is another important mechanism by which anticancer molecules exert their effects [16] . - [Mao et al (2019)] .  The arrest of the cancer cells at G2 M phase prevents the cancer cells from entering mitosis and as such the molecules that trigger the cell cycle arrest prevent the proliferation and tumourigenesis of cancer cells (Hua et al., 2018). - [Si et al (2019)] .  Previous studies have shown that depletion of cyclin A and B1 and upregulation of p27 and p21 results in the G2 M arrest of the cells during cell division [21]. - [Zhou et al (2019)] .  The arrest of cancer cells at G2 M phase prevents the cancer cells from entering mitosis, so the molecules that trigger cell cycle arrest prevent the proliferation and tumorigenesis of cancer cells [20]. - [Tan et al (2019)] .  Cell cycle arrest caused by several anticancer agents is an essential mechanism in the growth inhibition of cancer cells [19] . - [Wei et al (2019)] .  Furthermore, arrest of the cells at different phases of the cell cycle is an important route by which anticancer agents halt the growth of cancer cells [18]. - [Zeng et al (2019)] .  Besides autophagy, cell cycle arrest is another mechanism by which anticancer agents exert their anticancer effects [19] . -------------------------------------------------- Another list of papers cite Hua et al (2018) as precedents for methods which those authors did not in fact apply. - [Zhang et al (2019)] .  For determination of the ROS and mitochondrial membrane potential levels, the A549 cells were treated with 0, 9, 18, and 36 u00b5M concentrations of murrayanine for 24 hours and then the ROS and mitochondrial membrane potential levels in the A549 cells were determined, as described previously [11] *(in fact Hua et al did not measure or even mention ROS or mitochondrial membrane potentials)*. - [Guo et al (2019)] .  Plant-derived molecules have been reported to affect ROS levels in the cancer cells [15] . - [Xu et al (2019)] . Hua et al appear in the References but not in the text at all. - [Du et al (2019)] .  **Osteosarcoma** causes significant mortality world over and the treatments available are not efficient and are associated with a number of side-effects [11]. - [Yu et al (2019)] .  Several of the natural products have been reported to induce apoptosis and autophagy through production of ROS [18]. - [Liu et al (2019)] .  The colony formation assay was carried out as described previously [15] *[Hua et al did not perform colony-formation assays]* - [Zhu et al (2020)] .  Estimation of ROS was measured according to a method previously described [8]. - [Du et al (2020)] . Hua et al appear in the References but not in the text at all. ,