Clinical data
All subjects’ baseline characteristics are shown in Table 1. There were no differences between the two groups with regard to age and smoking history. As expected from the selected criteria, the forced expiratory volume in 1 second/fixed vital capacity ratio (FEV1/FVC) and forced expiratory volume in 1 second (FEV1% predicted) in COPD patients was significantly lower than control group (FEV1/FVC: 73.65±11.59 vs 94.87±11.13, P<0.01; FEV1% predicted: 60.52±8.75 vs 79.13±7.04, P<0.01). Although no hypoxemia existed in all patients with operation, the partial pressure of arterial oxygen of COPD patients were markedly lower than that in non-COPD patients (69.3±6.2 mmHg vs 82.6±5.2 mmHg, P<0.01).
Previous report and our recent study have proved the pulmonary vascular remodeling in COPD patients. In this study, we firstly evaluated the pulmonary vascular remodeling in COPD patients. As shown in Figure 1 (A-B), the lung tissue that was HE stained in each patient was focused on intermediate vessels (50-500µm) for evaluating pulmonary vascular remodeling. The pulmonary vascular wall thickness and muscularized vessels ratio in COPD patient were both significantly higher than non-COPD patients (Figure 1, C-D).
Circ-bptf Was Upregulated In Lung Of Copd Patietns
We screened 3610 expressed circRNAs by high-throughput transcriptome sequencing of explants of pulmonary artery from lung tissues of COPD patients or control and 48 differentially expressed circRNAs based on the changes more than 2 folds. Overall, 26 of these circRNAs were significantly upregulated, and 22 were significantly downregulated. We selected 4 significantly upregulated expressed circRNAs and we further verified the expression of the circRNAs by quantitative polymerase chain reaction (qPCR) using the pulmonary arterial smooth muscle strips from lung tissues of COPD patients or controls (Figure 2, A-E). Our results showed that circ-BPTF was the most significantly elevated circRNA, consistent with the sequencing results. We then chose circ-BPTF for subsequent analyses. Additionally, we performed fluorescence in situ hybridization analysis to determine circ-BPTF distribution and found that circ-BPTF was mainly colocalized with pulmonary arteries (Figure 2F).
Table 1: Baseline characteristic of all patients.
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7SMKZ41UVQKZ6au1Xh7GjDcX5pDOVB6o6JjnfzqPvXItltbOYcz7E0xigX3X90/h7pfdV2fitQfGb9uk21O2az8XSdzo84d1ZzpblXv5XReL4+n2VnvFuhufEate6YrforNnXt6vzoy+21WM6oXsDJoReaJa6CUJsTyAnlNqJ+9R55nYPs++mZxMHWte6ne/s6vUOdQH329ZhzLOs8J/VxwuvP09hq7pxruk/NA29WuSmJjtfFJ12+duMmPbfu4XzpxjXH5KfIuCu2evdZfBQ/tRGv2TSbn9l4Ol7zIjl3nF8jep+dvJ2Np+d3Xmis2XPdmm7Ou3m0VX+dr9fOxrsW01WvCc8kVgC6ZPFxJ4heWp9N5zMQ+nm0OHSuS9wumO6nP3W/fAbRcX/W/eqic/JjzXOcc6DPOVd1jvPciuYir+82UM/X//7v//75c9bU18+sP033GOWeOZ9Gm7TpfI790+j9FaeRXH8dnc85l27eLcfz3M5acu7oz5nVM9tqPD/D6n63Ru9Uc76bR1v115/qkzHX/I/GuxbTqqEkrpuHXnKV7E4qB0OB0ufaugQdTUIdU5ORSdldp2NqI3VMjHUbmjcvxa/Oh+ZiFvuqjt3dbzRfOl4Xp2kcXZNttig1tp7b7zbq634/neYo590Unzp/leJX49vNu6zG03U1L5Jzp3tW05xrnB2z8TyO3k19Rrl5i7r36ebRdvrrs/plu/aYTQunErW+kFtSwuqYk6gmv36eJWyaTUKeq32665zcbnVh+fxsweE/ug0t49clv9tq7jVvo7lJWkz1mKjvzkLbme88rz/1udtM9b74z9x186uYdXFLOl/jqPG62K7G07nZvHruu2cVXVv3j5nRePqc68Q5O7rvrdHc1HnQ+47m5rS/jtd95hoNC6cmvEskJ0zdqPTCOu6WSdwFb2S0cCSTtY6na0aJ7w1QLXk8rDlWOa+z+M3mMWkeR0VPY+sepr6jnNwpnLMFK97kkvrX99Dn2Sb9k4w2OcUo567T5Y+u6+ZyNZ7maTYnvtdojNPNejRel/f6PMu7W9KtB8VhFPuT/jo2m6NrMqwas2AoEDpvetFR0RJvSBmQUaKr7yyJfe96rY7XZ9Ax9/OkpG5SMVY3Bf082hC6DaTStfV6Xec5y/nx/OlP/zxrmWv6WcdmOSoeN5+pvqM+14W9es97pXh2RU5x3C1EGd/R2u/G854ya8k5UOdOal/db5Uro/G6vW5031uV76M4rWK10185oH5dPl2jfxXOuinVF1Gi1/OjJM4NxYFxy82o0vlRoul4DbwWlcet98xzdUz1vZWJuhY5/7M51BzlXEjOj857nGx1brPfKCdGMueU15XG07l8Dx9zy3OZS9l+Sg5lPGfvrX4ZN+viLRnXzmi8XTXXnGN1r8vmfNF99TlzbzSe+Rq307y9dhm3usa7eM36ez+pX4yuXZ+phxSkLjm0sLoNa0XX1QCbzn1GImqMW5ssAMD3+5TCWb9h2Kj47dA3l66wfWRMU/HVMwMAcOrTfuP0r+LZPkrj6q9B8q9G3vMbbNKYn1F8AQA/E792AQBwgMIJAMABCicAAAconAAAHKBwAgBwgMIJAMABCicAAAconAAAHKBwAgBwgMIJAMCBX/6/sqPRaDQajbbR3gooAADYQOEEAOAAhRMAgAMUTgAADlA4AQA4QOEEAOAAhRMAgAMUTgAADlA4AQA4QOEEAOAAhRMAgAMUTgAADlA4AQA4QOEEAOAAhRMAgAMUTgAADlA4AQA4MC2cj4+Pf/+L18/Pz3+9vLz89fv377ezn09j617Aezw9Pf1pgGnP+vtf7f//9vr6+nYGX0Vr8j3xd/15eHh4O/IfrhNuqk1fbVil9LL5wHo4PeSlCmcGAzjlDZLCCdMmrc3XvBFTPL9OfplV3Hfi77Wcc5euYY0Pq5QevBZJfb5U4RQXT+CUFpMWGoUTVvcqb9yX3MPwX12hzELacdEc/RapuVOf7zasUvpts/6KfGkUTryHFpkWJ4UTKxTOr6M4d3/NOqsrOjdbwzqvOfzuOjG8u4uY2qj66yV0zn+Nq6YNbHZtnlPLbw8+Z9nP8l45AflNpZ7D/dK8O8conFjR3pC/AeFytBbrX7fWPT7VPbzbxz13Gtt9v8O0bOsh/QJqemnLyu+H9zFvXg6A6foMhM+7eGZQ9XMNuvpnoHRezQHPe+NnyPmmcGJGe0ruH7gsrcWTwul64Dly/RmtaY/1HV+EpoXT/ID5UqIimJ9roGqQ1F/HkoufuL/G7IKVxTqbuHjybfLnUJ7kfFM4MZNf2nF5p4Wz6681Puov6l9rylcYPtGocGXy6eeTwqmf60vqGo/p/m6Vjo0KI4Xz51HeZL5kIw+Q6oaMy9N+nvVCVC/qMTsttKLxak35CsMn6l6uvoT6nBbOWpDzmuyvYzWIut8oSBROKF9qfgHKCf/nIHwd7cV1T9ZcZM1ItV6Ijo0KrWjNf8eePy2c9YH1OTcmfT4pnOqrz1n89NlJXfvr57yfr68TIRROUDhRKR9yv5H6hRyXo/jXPXqmrmHVmDp/pnowKsKXNnwLPZC/MbjlC9XjemF/dkHNPi5o9biLZtc/Pzt4ulc97oLrxrfLn4nCiZR7Urbv2mx/qpyH/MXGe3xds6of7p9Fs+7z3zmP8/IPAAD+gcIJAMABCicAAAconAAAHKBwAgBwgMIJAMABCicAAAconAAAHKBwAgBwgMIJAMABCicAAAconAAAHKBwAgBwgMIJAMCBX/nPtNBoNBqNRlu0twIKAAA2UDgBADhA4QQA4ACFEwCAAxROAAAOUDgBADhA4QQA4ACFEwCAAxROAAAOUDgBADhA4QQA4ACFEwCAAxROAAAOUDgBADhA4QQA4ACFEwCAAxROAAAOtIXz4eGh/1ev39rr62t7PNvv37/bfi8vL293+evPz3kuPT8//zmmMUaenp7efrosP0tHsfIz+j1Gz6Xj+f6jMa+d41FbfTc35cIu9e3GkxxTcX8PXff4+Pj26Z92ci7VdZIyRqP73bLd9/MesIppzamUOZHtJK9MzzrKHe9Hu+PO+mut+znvcf53eY3srtdV/9M5upThzu1krQmvBPcxJ3ull9M5czA66luD5P7d/U3Pp2svLRd08obgZ9Di8DvreJ1Yfa7H6hi3IufWMj45n6M86njeu766584Yoy8tirHGVus2sp2cS7P807NmDHS/e9o8T95Px1cxrTmizzl+l2863405mn/19fzm2JaFrq7Tzqy/njePqU/3DvdOcXYc6px2Vv1P5+iSjgtnUjKoz4o3rW6j0bHu+Or+owVyCd17alLzuXXek6nNIp9P7zB6Xi/oW6HnrXOiONT3TaO5T4rnbGHtzveq32yT38l5m92n5obneBWDW7H7foqn184spoplzonHszquzo/mcDX/Oj/KM9/X63hl1L++q+65eq57kPFQq3HW51Fsd/ufztGlHBXO+q2pKyhK8m6DUBC65KljWnd/07HRdZdQ31Of68LNydS5fNfVotF4t7yw9OyjRNZcjTY5q/GtfD5jPLKKo55l9DyznEvup6ZnS7pWx+saUP7Xvrfo5P00F+q3immdf/Wtm2hS/1EsV/Ov86Ox/W6rHLPd/sq32fvfOsVALeOqONe56I7Zbv/TObqU7cKpP+tL1IQXHesKZ9dXRgug3j/pmnwu9dM9NXH6Oe/j875/nsv+9Tn0rnmuXlcnTovDY6ivz+tYF4/kzeVWzZ59tEkl9VG8cz4yZp5rx2k2Zs3R6jMKZ809Nct8S3rmeuwW7b6fY+w5W8VUfTyvs/kVnR+Nt5p/51nH71bX9shOf91vtf5vkeZX767WqfkgisVo7e32P52jSxnueN5EstWk1IvWPmpdoviFMzj6ebQAZpuYg+kx3dxXP6tPnq8TkIvP/TwZdcI8hrhvfce8l+OkPn7fjGc36d2Yt0DPXPNCav441pXj5vkS/axjIzrnuGoefY+uZb6Jxq65YLOcm9EzZAy65++e5Vat3k9xdC7rz52YOg9WfXUu58/PMmo1N/VZ89XxM3Trs7Pqn89xL3Pv+VztVbuF0Hb7n87RpSwLp5NYf9Yk1IuqT9KxUVBr0tbxUr2/6XMG2IHMe+a13XlPfm16HvfXn5bv6Wvz/Ei+n67RtaPrdey7k+E99I6z53bsRnPdxcNzMBpXx0cLcJZToutG12benNA71M1YnzVWNvW7F7P3yzkY5XulPPG85liV+uX6r1bzr/N1rmyVd9VOf/dRuyeep9G8KsZ1nhT70drb7X86R5eyXTilvpg+14RQwo+S3otIY+vn2QLo7i+6Jo85kHlP30fnuvMaezSBea3le3bnO5p096nX6Oc68d2xW+C4zCgWap1RPGfxUN/R/I3uY7pudO0o53aMNmPpNoB7ku/nGHZtNDe6JuPj9dbNg+I8m5/V/Ov8VxZOcY7fI8VS71bfT3Guc6E5Hs3Pbv/TObqU4Wx6AcyStCucpnNKmEqBdkBmY4/uPwpk3svXSnd+lsg+l/3zPbvxKt0/J9Zj+l30c56X1ZjXSM+bG96I4qc2choP9R2NV/Oj0vOOnlnj5jzt0nOO7lvn/t6s3m/n/bsNUvtEzQmNMSp6tpp/nf/qwrnz3PdAsXA81Oo76/NsTe/0P52jS7lI4dTLjhLF16w23O7+GrcGzIHMBaN7e2P1+ToB6lOfwWPoXD6/ftYYav48m7hu8foZ1PRzfS+PfUv0nqsEdvxnPNemuRvlh8YbzU3XaoHVuKOxu5zboWs6io/O1dy7Fzvv1+V75T3BfZwz9Rr16+ZTfUetrkV9zvxJvu8qp223v55xd8x7kvvkbE3bTv/TObqUdsXXzagmn+T5rtUETzo/e/G6GDxW9xwOpBexfxYvWrd6zzynlvK4x7ZZEtRxLJ+lPofG697t2nXvWmPebVKe36QY+JoaWx9XG216K86TbDpmo5wTz7/71/VR+fwoR27dyft1hbPGU3zMLc+Z7tsd35Xjq+nZLPNPLd9N61XHct3O+tf8yPv8NI6BYpK6mMqov8xi/tX6Xf6GaCEpiF+dnJrYz7qnnv8jGwIA4OtQOD9A963fmE5857MDAN7npgunCo4Kj9tHith76a8L3vvbop4ZAHBb2LkBADhA4QQA4ACFEwCAAxROAAAOUDgBADhA4QQA4ACFEwCAAxROAAAOUDgBADhA4QQA4MCv/L+so9FoNBqNtmhvBRQAAGygcAIAcIDCCQDAAQonAAAHKJwAABygcAIAcIDCCQDAAQonAAAHKJwAABygcAIAcIDCCQDAAQonAAAHKJwAABygcAIAcIDCCQDAAQonAAAHKJwAABxoC+fDw0P/r16/tdfX1/Z4tt+/f7f9Xl5e3u7y15+f81x6fn7+c0xjjKye8/Hx8UPPUM9pPPGfSX19PK9THDp6dstrb5neQe+c7yZPT09/x2M2n6aYub+acsHqnOyMV9WcGM2RZV+3Ol/OV7fVmPck39st41PjvTNnvibn3jLWNdd21Bxy6+6V3G+1VtVH97hHuzGwVX+v9VuL1/A3Tr9QTXIll485gSsFIZPQBa6jvjX53b+7v+n5HGxNSjcx2rDt9Bm82eeE6mcdq/fSu+b1vs796zto7HrM8b5Fo7iI3tXzoHfu4lHlvCVdl/fQz6PxMj8qXZP5qc+jvhqnjq9r83oZPfO9W8VH5zK+zvNuzkzXqk+dJ9H1eUzrbhR79evuU8cU5dIoByTfQdd3uS56lux7T3ZjYKv+Xr+3GK/jwpkUDPVZUVBGwdGx7vjq/rlYNAGrSTx5Bt971DeLZPc5n1s/azzTz/k56fjqPa6N37fbjBSDjIVo3mZFRjHo4i41bh6/i+donDo/oph3Y0g3hvrn9Xr3/PyTrOKj2NT51vlZDliXVzXOOj9aM6N56Z65fnFO3T30bHUcfVbf7twt0jv4XXZjYCcxu8V4HRVOBSM5SZIC0AVh9M2wjmnd/U3H8jpNUE7SR59B/WYLKcdQv9x0dd98bv2c51cbht5jtIlfo1FMRe9R49gdSzqnmNW8GqnxNR3rckDzUq9RzLu+HV1fn9/PO3uvn6LGR7Gt+aH1thMrxXS0P5jGHs2drvU6nFEuzNal3qE+R3dMYzi/dvPp2vj51fSOthsD2+3v/fLW4rVdOPVntwDUJ+lYF4Sur9RAWr1/0jV5XBOiZh95BifObCGZJ73SMZ3zeT9rPuOInmWn3zXw+zmuarkhKob1XTyvI46VrvXYM+rT5Yju0+WAeGyd1/jqu0v985lqHmrck/HuTY2PYlKLpM5/RuHUuVmsdb7LjUrPOMoV6eZU1+QeoZ91LzXn1i3RfOi5R/OyE4O02997yK3Fa7iD6aX1QtnqSysxax+1LghOqFwIs8T2/bvzmoCkz3l/tfc+gydylBBJ13aJlrFz8qivn0lj+3x9P9//Fuid9KyOp+Pr2OnPOleOzY5ZDojO+96O26zlOM4ZX79L142eRxyTn6rGx3PodSDKi48WTm/0as4332vWOqtn0XX5/KL39H2Vez7vNeC1fu0cs1lOyyoG1W5/r9tbiZctC6cDqj/rS3ebhI6NgqDrM0lHQZfRhOpzXUyaEDX7yDN4ImfPZuqT9x3JZ/b4omN10fp8fe9r1L2/3snv1533vO7S9XUB2mzD0zWjHFBsPe7uXIuuW22yoj6je9+zUXycE9l2Yq5+XjcjHq9bL7p2tY6UA6tn0fg1B5U/vi6v1/3U/9bmX/Om5x5ZxaDa7e/97tbiNYyUN5VMvJrEuUmaAjAKgoOksfXzbFF095duMWhC1Oyjz6DzOd6IkmCnX/bR/by5+Fnyfbpj16p7f8+bf64bab7/DvXXONUq7p7fTsZXf+pzlweV+uz0U1xG975nO/E5ye+defH8dePp2tV9lEeruVKf+hzKYR3z+3RtlaPXyM9e1+gsBp3d/o7fra2Xo8JZKQjq09G5LhgKnoKqzWU29uj+9RuLaLyTJF09g99rNJm+Tv1qglX1HnmNkybP+9gt8BwlHfP76b3q+ykeisEuzVNeLxpjtdBGhbN7Zj3PTv6oz+q+ssqJe7UTH8Vmd/41Tzt9R+tF19bcqUbXpi4/al6bc34nT66Z38PvchID2e2vsW8xXsOsGRWupOCoT6UgjDYPX1ODWnX317g6Xmms1Xhp5xl0Tn3q/XTcxzzpI93z5jV6jhqnLuGumZ5Vhcz0PvnOOufzq3hVioVa0ljdnHjsWVMuqennHFfX1/tUum6nIHbP9xOs4uP5yVxZqfPU0Xjuo7h7rkctqf/u8+havYPomtF1zi/3vSe7MbCd/s6LW4tXu4tpAehl3LoXzvNdmyW8zs82F21k3Vj1ORz0bLsToL6rDU73XY1fC4Wp36gA5rhaaEnXrJ7r2mS+dM+e85nv603G86pr3U+t5lDNi1G/lZo3eb3P1TH1ubuPjuVYtzZ3n2UnPjXXpYt3zYMsyDUHPhJvjVXXs/iZ85xzVU3XjbhfN+6tm8XgPTHTuvd5ta7OXKv9r/9oKVFm37RPfOZYAIDLoHB+An9D/ojPGAMAcHns1J9Efy0x+yucGf2m+d5rAQBfi8IJAMABCicAAAconAAAHKBwAgBwgMIJAMABCicAAAconAAAHKBwAgBwgMIJAMABCicAAAd++f+Znkaj0Wg02kZ7K6AAAGADhRMAgAMUTgAADlA4AQA4QOEEAOAAhRMAgAMUTgAADlA4AQA4QOEEAOAAhRMAgAMUTgAADlA4AQA4QOEEAOAAhRMAgAMUTgAADlA4AQA4QOEEAOBAWzgfHh7af/X66enprce+379//2ucj3p9ff3zjF/h8fHxH8+uz5bH1fSuM1/53Nfs+fn575gpJpaxVp8ZXee+ai8vL29n3sf3ns2P7pH3zGfH58j41hz4SPx35ndE+17ed5Zrmds/ea3nWt6x6u/1vtoXvsrwrVzwnJz+rBfc5WAkJ9aqyIz4Ob6SF2y3YPQ+JwvEY/3UTVfv3s2fciW/mKnPbJFkzJ1THcV5No7nY5XXGif7OLd/6jxeguY0c0CfvU+8N/4786t7dGtbVvmTNE721X1/YvFUrB0Hx39m1V/ndExtdy4ubfhGLlCZmLvJKv6W1vV1IHbGSeqv676aJ7NbXHqX08WhcX7iglIMuw2sm9fZF6SaN76+yycdGy02z+vOYvQGbr5nPY73cTxzjWkP8dy8J/6786sxurUtWchX6rPos+7f5eU9yblwzFMWxuqk/85cfpVhFeomXQ+tY6MkMwdylHSr8yOzCbgkT2733nqe9xTB3BR+Ar3vKE7OteQcWeWaqM8s10Zx1vOc5mDS89XNEu+neOZcaW5m8V3Ff3d+NUaXZ85Bte4L34r3DY1zjxybXNeavxqr7pid9Ne9Mj++01HhVBLmMX92cxI7YWYvqWA74L6X2mijdBLXJNQ9NI7P5339OSfW/XQfTU6ez/fJ5/D7dM/m+6d8n2x5vfrU6+6Z3l+xynh4Lh2vjE/O04z6jRal6HyXh57TfKbT+dA1fgd8nOdC+aDWzVuaxf9kfnWvLs9ybF2rMVb5mNT33ta490y1js7XLyuag1EcTvrrnquc+CrbhdOJ6E3Kn31eL++X9bWzl1Rf93fgdN0owB4zdYsij4meTz/rev/spnfIY+ojeke/p/hdRy2f2X31p/h5HCfzfevxe+SYZExz/qWer3Hs5FyrWR7rmubZ1+pP8XzURTziMfC5FP+deVjFfza/zq1Z69alrh3tTx3f6x7srEfRGq5zpzkYxe2kv+4/m/OvtCyc2WYvn+cd5NlLqm833ugeo2DW4753Jr4+uyh6AWUC6Pp8Vk3k7iZe7++4WXc/GR2/RzUmUmPqeNS2Q/Olvl2+adzueJ1jUb/de47yFB+juDo36vykVfxP5lf5ubsOdb1yakXj1YJwD7zXj2Kg8/W96x6ZTvrrvt1a/g7DXcKb3SxJHEQlSb6sN8GauObzXWKNrhkFsx73osvn1uevKpyS9+uKhnTPca+6GKzev87Jiuar66/7dMfrHMtorqpRjuJjNOeZD5qLkz0incyvju+uQ42Ze8vIveeI5kqxrPHUWqvv3s2FnfTXvU72hEsa7hJOslGS6AWyYNTPevnR9erbnZsl8Cjp6311fR1bn3W96Lg+5310fU5InTiP2T1bvb/4Wd266/wcXXzuTRfz2ft3MV3RnHmOk8bPubUun3RsdV/dp5tPfIzXWOZDNx+78T+ZXx3fndOdvDzN3VunOKs5jjXu2kt1rnPSX/26tfwd/l2J3ujB9aDdxiZ6gXxhvWxNGH2uQfF1XWC0KEZGG23dZLsFmPfzOLlQdH1OyEcKp8bPa0f0PDVe96zGSTHu5lt9FOsTmpccW9fPmnNB85TPoDG6vDT1red35hp7NDc5HzVHTuM/ml+v51mr+4xovG4PSLpHvfan5Yje1/up4p1rs7PbX/OS+/R3ancoPXgmUSZfqn38c9KLZr963vL4KDgZYMl7qinos8+r/jqve/iz4pCf1fTZ8riaxhstypoMute1JMFXyfjrZ/OXGbVRTBQ/x7DOW87Jqcx1jWt+Jj9nzQO3nzaHl5R5oJY5sop/nS8bze+OzFe1WjSdhx43+2ZbFdt7lHFP3h/ruhn1l7revQ98p76KfaG6WNR0rOO+10zP2C1QJYwX0C28BwCgd3O7t7+xjIrrd9O3oa5w+reia39+AMDczf7acw2/rnf822RtOq52rc8NANjD3xcCAHCAwgkAwAEKJwAAByicAAAcoHACAHCAwgkAwAEKJwAAByicAAAcoHACAHCAwgkAwIFf9f8ajkaj0Wg02qS9FVAAALCBwgkAwAEKJwAAByicAABs++uv/wOqJho6gLdo/wAAAABJRU5ErkJggg==)
Circ-bptf Regulated Hpasmcs Proliferation Exposed To Hypoxia
Next, we investigated the role of circ-BPTF in pulmonary arterial smooth muscle proliferation under hypoxic conditions. Hypoxia induced circ-BPTF upregulation in hPASMCs in vitro as a time-dependent manner (Figure 3A). Specific siRNA targeting circ-BPTF significantly inhibited mRNA levels of circ-BPTF in hPASMCs exposed to hypoxia, and we found that siRNA-1 had the most obvious effects (Figure 3B). Therefore, we transfected hPASMCs with siRNA-1 to study the function of circ-BPTF.
As shown in figure 3C, the results of CCK-8 indicated that hypoxia induced proliferation as a time-dependent manner. But siRNA targeting circ-BPTF significantly inhibited proliferation in hypoxia exposing cells. This results were also proved by MTT assay (Figure 3D). We then analyzed the expression of PCNA, a widely used cell proliferation marker. By results from immunocytochemistry staining (Figure 3E and 3F), we showed that hypoxia-induced PCNA over-expression were reversed by knockdown of circ-BPTF following transfection of PASMCs with circ-BPTF siRNA. Additionally, hypoxia decreased the percentage of TUNEL positive cells, which was also reversed after knockdown of circ-BPTF (Figure 3E and 3F).
Circ-bptf Adsorbed Mir-486-5p
CircRNA acts role as a competitive endogenous sponge for miRNA. We speculated that circ-BPTF might serve as molecular sponge to promote PASMCs proliferation. Based on the bioinformatics software (miRanda and RNAhybrid), we predicted that circ-BPTF might adsorb miR-486-5p, miR-202-3p, miR-758-3p and miR-218-5p (Figure 4A). After exposed to hypoxia, qPCR results showed that miR-486-5p and miR-758-3p levels was downregulated, but miR-202-3p and miR-218-5p expression levels was upregulated (Figure 4B). The change of hsa-miR-218-5p was the most significant. Moreover, a recent report has demonstrated the downregulation of miR-486-5p in PASMCs. So, we focused on miR-486-5p in subsequent analyses. We predicted that circ-BPTF and miR-486-5p had 2 binding sites by TargetScan analysis (Figure 4C).
Next, we used a dual luciferase vector plasmid system to verify whether circ-BPTF bound to miR-486-5p. We found that the ratio of luciferase for wild-type circ-BPTF was significantly decreased when compared with that of the negative control after miR-486-5p exposure (Figure 4D). However ratio of luciferase for mutant circ-BPTF did not change as compared with the negative control after miR-486-5p exposure. Moreover, circ-BPTF specific siRNA significantly reversed the suppression of circ-BPTF on miR-486-5p expression (Figure 4E). These results indicated that circ-BPTF could be used as a molecular sponge for miR-486-5p.
Circ-bptf Regulated Cell Proliferation By Adsorbing Mir-486-5p
To indentify the role of miR-486-5p in cells proliferation, PASMCs transfected with miR-486-5p mimics and exposed to hypoxia for 24-72 hours. The results of CCK-8 showed that miR-486-5p mimics significantly inhibited hypoxia-induced cells proliferation (Figure 5A). The PCNA positive cells were also significantly decreased by miR-486-5p transfection via the results of immunocytochemistry staining (Figure 5B and 5C). Additionally, miR-486-5p mimics increased the TUNEL positive cells percentage (Figure 5B and 5C).
Next we used recovery experiments to identify whether circ-BPTF promoted proliferation via adsorbing miR-486-5p. We co-transfected PASCMCs with circ-BPTF siRNA and miR-486-5p inhibitors before exposure to hypoxia. The results of Edu assays showed that circ-BPTF siRNA or miR-486-5p mimics both inhibited hypoxia-induced proliferation, whereas miR-486-5p inhibitor could reverse the inhibition role of circ-BPTF siRNA on cells proliferation (Figure 5D and 5E). These results demonstrated that circ-BPTF could regulate PASCMCs proliferation by adsorbing miR-486-5p.
Mir-486-5p Targeted Cemip In Pasmcs With Hypoxia Exposure
Previous reports have demonstrated cell migration-inducing protein (CEMIP) as a down-stream target protein of miR-486-5p, we next try to identify the regulator role of miR-486-5p in CEMIP expression in PASMCs. Based on the bioinformatics software (miRanda and TargetScanHuman ), we predicted two putative binding sites for miR-486‐5p on the 3′‐UTR of CEMIP mRNA (Figure 6A). The results of dual luciferase assays showed that ratio of luciferase for wild-type CEMIP was significantly decreased when compared with that of the negative control after miR-486-5p mimics exposure (Figure 6B). However ratio of luciferase for mutant CEMIP did not change as compared with the negative control after miR-486-5p mimics exposure. These results indicated that miR-486-5p could regulate CEMIP expression.
Next, we explored the regulation role of miR-486-5p on CEMIP expression. As shown in Figure 6C, miR-486-5p mimics markedly suppressed the mRNA levels of CEMIP in human PASMCs after hypoxia exposure. Furthermore, miR-486-5p mimics significantly inhibited CEMIP protein levels, while miR-486-5p inhibitor raised CEMIP protein levels (Figure 6D and 6E). These results indicated that CEMIP might be a target of miR-486-5p in hypoxia-induced PASMCs proliferation.
Cemip Promoted Proliferation In Pasmcs With Hypoxia Exposure
Subsequently, we tried to uncover the role of CEMIP in hypoxia-induced PASMCs proliferation. Figure 7A showed the data from GEO database. (GSE56698). Microarray study from lung homogenates revealed that CEMIP is highly up-regulated in hypoxic mice in comparison with those from control mice. Next, we further examined the CEMIP expression in human PASMCs. As shown in Figure 7B and 7C, hypoxia exposure not only raised CEMIP mRNA levels, but also promoted CEMIP protein levels. Moreover, hypoxia raised CEMIP expression as a time-dependent manner (Figure 7C).
Then we used CEMIP specific siRNA to knockdown its expression. In PASMCs exposed to hypoxia, specific siRNA significantly prevented CEMIP expression (Figure 7D). Although hypoxia raised Edu positive cells percentage, CEMIP specific siRNA significantly reversed it as compared with negative control siRNA (Figure 7E and 7F). On the contrary, CEMIP specific siRNA raised TUNEL positive cells in hypoxia-exposed PASMCs (Figure 7G). Additionally, CEMIP specific siRNA prevented the protein expression of p-AKT, cyclin D1 and cIAP2 (Figure 7H and 7I). These results showed that CEMIP might promoted PASMCs proliferation via p-AKT, cyclin D1 and cIAP2 pathways after hypoxia exposure.
Circ-bptf Regulated Mir-486-5p/cemip Axis
To identify the regulation of circ-BPTF on miR-486-5p/CEMIP Axis, we co-transfected PASCMCs with circ-BPTF siRNA, miR-486-5p inhibitors and inhibitors before exposure to hypoxia in the recovery experiments. The Western-blot results showed that circ-BPTF specific siRNA and miR-486-5p mimics both could inhibit hypoxia-induced CEMIP protein up-regulation. But miR-486-5p inhibitors reversed the inhibitory effect of circ-BPTF specific siRNA on CEMIP protein expression (Figure 8A and 8B). Circ-BPTF specific siRNA suppressed not only the CEMIP expression but also its downstream target, p-AKT (Figure 8C and 8D). These results demonstrated that the role of circ-BPTF/miR-486-5p/CEMIP axis in regulating hypoxia-induced PASMCs proliferation.